Methods and compositions for treating SERPINC1-related disorders

Abstract

This invention provides a method for treating patients with bleeding disorders such as hemophilia. [Solution] The present invention relates to an iRNA, such as a double-stranded ribonucleic acid (dsRNA), composition that targets the Serpinc1 gene, and to a method of using such an iRNA, such as a dsRNA, composition to inhibit the expression of Serpinc1 and to treat subjects with Serpinc1-related diseases, such as bleeding disorders such as hemophilia.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 264,013 filed on 7 December 2015, U.S. Provisional Patent Application No. 62 / 315,228 filed on 30 March 2016, U.S. Provisional Patent Application No. 62 / 366,304 filed on 25 July 2016, and U.S. Provisional Patent Application No. 62 / 429,241 filed on 2 December 2016. The entire contents of each of the aforementioned patent applications are incorporated herein by reference.

[0002] This application relates to U.S. Provisional Patent Application No. 61 / 992,057 filed on 12 May 2014, U.S. Provisional Patent Application No. 62 / 089,018 filed on 8 December 2014, U.S. Provisional Patent Application No. 62 / 102,281 filed on 12 January 2015, and PCT Patent Application No. PCT / US2015 / 030337 filed on 12 May 2015. The entire contents of each of the aforementioned patent applications are incorporated herein by reference.

[0003] Furthermore, this application relates to U.S. Provisional Patent Application No. 61 / 638,952 filed on 26 April 2012, U.S. Provisional Patent Application No. 61 / 669,249 filed on 9 July 2012, U.S. Provisional Patent Application No. 61 / 734,573 filed on 7 December 2012, U.S. Patent Application No. 13 / 837,129 filed on 15 March 2013 (current U.S. Patent No. 9,127,274), U.S. Patent Application No. 14 / 806,084 filed on 22 July 2015 (current U.S. Patent No. 9,376,680), U.S. Patent Application No. 15 / 070,358 filed on 15 March 2016, and PCT Patent Application No. PCT / US2013 / 038218 filed on 25 April 2013. This application is also related to PCT patent application PCT / US2012 / 065601, filed on November 16, 2012. The entire contents of each of the aforementioned patent applications are incorporated herein by reference.

[0004] Sequence List This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on December 6, 2016, is named 121301-05220_SL.TXT and is 21,140 bytes in size. [Background technology]

[0005] Serpinc1 is a member of the serine proteinase inhibitor (serpine) superfamily. Serpinc1 is a plasma protease inhibitor that inhibits thrombin and other active serine proteases of the coagulation system, such as factors X, IX, XI, XII, and VII, thereby modulating the blood coagulation cascade. The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans that catalyze the formation of the thrombin:antithrombin (TAT) complex.

[0006] A bleeding disorder, whether hereditary or acquired, is a condition in which blood clotting is insufficient. For example, hemophilia is a group of inherited bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder with a functional deficiency of coagulation factor VIII and accounts for 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder with a functional deficiency of coagulation factor IX. Hemophilia B accounts for approximately 20% of hemophilia cases. Hemophilia C is an autosomal inherited disorder with a functional deficiency of coagulation factor XI. Hemophilia C is not entirely recessive, as heterozygous individuals also show increased bleeding.

[0007] Currently, there is no cure for hemophilia, but it can be controlled by regularly infusing the deficient clotting factor, for example, factor VIII in hemophilia A. However, some hemophilia patients develop antibodies (inhibitors) against the supplemented factor they receive, making them resistant to the supplement. Consequently, bleeding in such patients cannot be properly controlled. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] For example, the development of high-titer inhibitors against factor VIII and other coagulation factors is one of the most serious problems in hemophilia treatment, making bleeding management extremely difficult. Currently, the only ways to stop bleeding in such patients are the use of "bypass agents" such as factor VIII inhibitor bypass activity (Fiber) and recombinant activated factor VII (rFVIIa), plasmapheresis, continuous factor replacement, and immunotherapy, but none of these are completely effective. Therefore, alternative treatments for patients with bleeding disorders such as hemophilia are needed in this field. [Means for solving the problem]

[0009] The present invention provides a method for treating subjects with a disorder that would benefit from inhibiting or reducing the expression of the Serpinc1 gene, such as a bleeding disorder including hemophilia, and provides a method for inhibiting the expression of the Serpinc1 gene by using an iRNA composition that induces RNA-induced silencing complex (RISC)-related cleavage of the RNA transcript of the Serpinc1 gene.

[0010] The present invention is at least in part based on the remarkable discovery that very low doses (e.g., doses at least about 30 times lower than those taught in the art) of GalNAc-binding double-stranded RNAi agents containing specific chemical modifications exhibit extraordinary potency in inhibiting Serpinc1 expression, as well as an extraordinary duration of inhibition of Serpinc1 expression. Specifically, low-dose RNAi agents containing a GalNAc ligand and sense and antisense strands with substantially all of the nucleotides modified, such as one or more motifs of three identical modifications on three consecutive nucleotides, including one motif at or near the drug cleavage site, six phosphorothioate linkages, and a GalNAc ligand, have been shown herein to be exceptionally effective and long-lasting in silencing the activity of the Serpinc1 gene.

[0011] Accordingly, in one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject that would benefit from reduced Serpinc1 expression. The method comprises administering a double-stranded RNAi agent to a subject at a dose of about 0.200 mg / kg to about 1.825 mg / kg, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 5 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with the ligand, for example, the ligand is bound at the 3' end of the sense strand.

[0012] In another aspect, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a double-stranded RNAi agent to a subject at a dose of about 0.200 mg / kg to about 1.825 mg / kg, and the double-stranded RNAi The drug comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising the nucleotide sequence of SEQ ID NO: 1 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, and the antisense strand comprising the nucleotide sequence of SEQ ID NO: 5 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi drug comprises a ligand, for example, the sense strand of the double-stranded RNAi drug is conjugated with a ligand that binds at the 3' end of the sense strand.

[0013] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0014] In another embodiment, the sense strand and antisense strand include a complementary region containing at least 15 consecutive nucleotides that are different from any one sequence listed in either Table 2 or 3 and not exceeding 3 nucleotides.

[0015] In some embodiments, the modified nucleotide is independently selected from the group consisting of 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides bonded to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In yet another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-allyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0016] In another embodiment of the double-stranded RNAi drug, at least one strand includes a 3' overhang of at least one nucleotide. In yet another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, at least one strand of the RNAi drug includes a 5' overhang of at least one nucleotide. In a particular embodiment, at least one strand includes a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug include an overhang of at least one nucleotide.

[0017] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0018] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0019] In a particular embodiment, the ligand is as follows: [ka]

[0020] In another embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a double-stranded RNAi agent to a subject at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to the mRNA encoding Serpinc1, and each strand being about 14 to about 30 nucleotides long, and the double-stranded RNAi agent is given by formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by, n p ' is a 2-nucleotide overhang, n pEach nucleotide within the ’ is linked to an adjacent nucleotide via a phosphorothioate linkage; each N a and N a ’ represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, independently modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; YYY and Y’Y’Y’ each independently represent one motif of three identical modifications in three consecutive nucleotides, the modification being a 2’-O-methyl or 2’-fluoro modification; The sense strand and the antisense strand each independently contain two phosphorothioate linkages at the 5’ end; The sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker, thereby preventing at least one symptom of a subject having a disorder that would benefit from a decrease in Serpinc1 expression.

[0021] In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in Serpinc1 expression. The method comprises administering to the subject a dose of a double-stranded RNAi agent from about 0.200 mg / kg to about 1.825 mg / kg, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding Serpinc1, each strand being from about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent having the formula (IIIe): Sense: 5’-N a -YYY-N a -3’ Antisense: 3’n p ’-N a ’-Y’Y’Y’-N a ’-5’(IIIe) represented by: n p ’ is a 2-nucleotide overhang, and n pEach nucleotide within is linked to an adjacent nucleotide via phosphorothioate linkage; Each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two distinctly modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications in three consecutive nucleotides, where the modifications are 2'-O-methyl or 2'-fluoro modifications; The sense strand and antisense strand each independently contain two phosphorothioate links at their 5' ends; The sense chain is conjugated with at least one ligand, the ligand being one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. This prevents at least one symptom in individuals with a disorder who would benefit from reduced Serpinc1 expression.

[0022] In one embodiment, the double-stranded RNAi drug is administered to the subject in a single dose or in two or more doses, for example, three, four, five, or six doses.

[0023] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month, once every six weeks, once every two months, once a quarter, or as needed.

[0024] Double-stranded RNAi drugs are, for example, approximately 0.200 to 1.825 mg / kg, 0.200 to 1.800 mg / kg, 0.200 to 1.700 mg / kg, 0.200 to 1.600 mg / kg, 0.200 to 1.500 mg / kg, 0.200 to 1.400 mg / kg, 0.200 to 1.400 mg / kg, 0.200 to 1.200 mg / kg, 0.200 to 1.100 mg / kg, 0.200 to 1.000 mg / kg, 0.200 to 0.900 mg / kg, and 0.200 to 0.800 mg / kg. g / kg, approximately 0.200 to approximately 0.700 mg / kg, approximately 0.200 to approximately 0.600 mg / kg, approximately 0.200 to approximately 0.500 mg / kg, approximately 0.200 to approximately 0.400 mg / kg, approximately 0.225 to approximately 1.825 mg / kg, approximately 0.225 to approximately 1.800 mg / kg, approximately 0.225 to approximately 1.700 mg / kg, approximately 0.225 to approximately 1.600 mg / kg, approximately 0.225 to approximately 1.500 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.200 mg / kg, approximately 0.2 25 to approximately 1,100 mg / kg, approximately 0.225 to approximately 1,000 mg / kg, approximately 0.225 to approximately 0.900 mg / kg, approximately 0.225 to approximately 0.800 mg / kg, approximately 0.225 to approximately 0.700 mg / kg, approximately 0.225 to approximately 0.600 mg / kg, approximately 0.225 to approximately 0.500 mg / kg, approximately 0.225 to approximately 0.400 mg / kg, approximately 0.250 to approximately 1.825 mg / kg, approximately 0.250 to approximately 1.800 mg / kg, approximately 0.250 to approximately 1.700 mg / kg, approximately 0.250 to approximately 1.600 mg / kg, approximately 0.250 to approximately 1.50 0 mg / kg, approximately 0.250 to approximately 1.400 mg / kg, approximately 0.250 to approximately 1.400 mg / kg, approximately 0.250 to approximately 1.200 mg / kg, approximately 0.250 to approximately 1.100 mg / kg, approximately 0.250 to approximately 1.000 mg / kg, approximately 0.250 to approximately 0.900 mg / kg, approximately 0.250 to approximately 0.800 mg / kg, approximately 0.250 to approximately 0.700 mg / kg, approximately 0.250 to approximately 0.600 mg / kg, approximately 0.250 to approximately 0.500 mg / kg, approximately 0.250 to approximately 0.400 mg / kg, approximately 0.425 to approximately 1.825 mg / kg, approximately 0.425 to approximately 1,800 mg / kg, approximately 0.425 to approximately 1,700 mg / kg, approximately 0.425 to approximately 1,600 mg / kg, approximately 0.425 to approximately 1,500 mg / kg, approximately 0.425 to approximately 1,400 mg / kg, approximately 0.425 to approximately 1,400 mg / kg, approximately 0.42. 5 to approximately 1,200 mg / kg, approximately 0.425 to approximately 1,100 mg / kg, approximately 0.425 to approximately 1,000 mg / kg, approximately 0.425 to approximately 0.900 mg / kg, approximately 0.425 to approximately 0.800 mg / kg, approximately 0.425 to approximately 0.700 mg / kg, approximately 0.425 to approximately 0.600 mg / kg, approximately 0.425 to approximately 0.500 mg / kg, approximately 0.450 to approximately 1.825 mg / kg, approximately 0.450 to approximately 1,800 mg / kg, approximately 0.450 to approximately 1,700 mg / kg, approximately 0.450 to approximately 1,600 mg / kg, approximately 0.450 to approximately 1,500 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.200 mg / kg, approximately 0.450 to approximately 1.100 mg / kg, approximately 0.450 to approximately 1.000 mg / kg, approximately 0.450 to approximately 0.900 mg / kg, approximately 0.450 to approximately 0.800 mg / kg, approximately 0.450 to approximately 0.700 mg / kg, approximately 0.450 to approximately 0.600 mg / kg, approximately 0.450 to approximately 0.500 mg / kg, approximately 0.475 to approximately 1.825 mg / kg, approximately 0.475 to approximately 1.800 mg / kg, approximately 0.4 75 to approximately 1,700 mg / kg, approximately 0.475 to approximately 1,600 mg / kg, approximately 0.475 to approximately 1,500 mg / kg, approximately 0.475 to approximately 1,400 mg / kg, approximately 0.475 to approximately 1,400 mg / kg, approximately 0.475 to approximately 1,200 mg / kg, approximately 0.475 to approximately 1,100 mg / kg, approximately 0.475 to approximately 1,000 mg / kg, approximately 0.475 to approximately 0.900 mg / kg, approximately 0.475 to approximately 0.800 mg / kg, approximately 0.475 to approximately 0.700 mg / kg, approximately 0.475 to approximately 0.600 mg / kg, approximately 0.475 to approximately 0.50 0 mg / kg, approximately 0.875 to approximately 1.825 mg / kg, approximately 0.875 to approximately 1.800 mg / kg, approximately 0.875 to approximately 1.700 mg / kg, approximately 0.875 to approximately 1.600 mg / kg, approximately 0.875 to approximately 1.500 mg / kg, approximately 0.875 to approximately 1.400 mg / kg, approximately 0.875 to approximately 1.400 mg / kg, approximately 0.875 to approximately 1.200 mg / kg, approximately 0.875 to approximately 1.100 mg / kg, approximately 0.875 to approximately 1.000 mg / kg, approximately 0.875 to approximately 0.900 mg / kg, approximately 0.900 to approximately 1.825 mg / kg, approximately 0.900 to approximately 1,800 mg / kg, approximately 0.900 to approximately 1,700 mg / kg, approximately 0.900 to approximately 1,600 mg / kg, approximately 0.900 to approximately 1,500 mg / kg, approximately 0.900 to approximately 1,400 mg / kg, approximately 0.900 to approximately 1,400 mg / kg, approximately 0.900 to approximately 1,200 mg / kg, approximately 0.900 to approximately 1,100 mg / kg, approximately 0.900 to approximately 1,000 mg / kg, approximately 0.925 to approximately 1,825 mg / kg, approximately 0.925 to approximately 1,800 mg / kg, approximately 0.92 The drug can be administered to the target patient once a month in doses of approximately 5 to 1,700 mg / kg, approximately 0.925 to 1,600 mg / kg, approximately 0.925 to 1,500 mg / kg, approximately 0.925 to 1,400 mg / kg, approximately 0.925 to 1,400 mg / kg, approximately 0.925 to 1,200 mg / kg, approximately 0.925 to 1,100 mg / kg, or approximately 0.925 to 1,000 mg / kg, for example, once a month for 1, 2, 3, 4, 5, 6, 7, 8 months or longer.

[0025] The subjects may include individuals with acquired or hereditary bleeding disorders, such as hemophilia, specifically hemophilia A, hemophilia B, or hemophilia C.

[0026] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0027] In one embodiment, administration of a double-stranded RNAi agent to a subject results in increased blood coagulation and / or decreased Serpinc1 protein accumulation.

[0028] In one embodiment, the method further includes measuring the thrombin level of the subject.

[0029] Double-stranded RNAi drugs can be administered subcutaneously or intravenously.

[0030] In one embodiment, substantially all nucleotides of the antisense strand and substantially all nucleotides of the sense strand of the RNAi drug contain modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications. In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand of the RNAi drug are modified nucleotides.

[0031] In one embodiment, the YYY motif occurs at or near the break point of the sense chain.

[0032] In one embodiment, the Y'Y'Y' motif arises at the 11th, 12th, and 13th positions from the 5' end of the antisense chain.

[0033] The double-stranded region may be 15-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-25 nucleotide pairs long, 23-27 nucleotide pairs long, 19-21 nucleotide pairs long, or 21-23 nucleotide pairs long.

[0034] Each chain may contain 15 to 30 nucleotides, or sometimes 19 to 30 nucleotides.

[0035] In one embodiment, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0036] In one embodiment, the ligand is as follows: [ka]

[0037] In one embodiment, the ligand is bound to the 3' end of the sense strand.

[0038] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0039] In one embodiment, the base pair at the first position of the 5' end of the antisense strand of the double helix is ​​an AU base pair.

[0040] In one embodiment, the RNAi agent is AD-57213 ((sense (5'→3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf (SEQ ID NO: 13); antisense (5'→3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage)).

[0041] In one embodiment, the drug is administered as a pharmaceutical composition. In another embodiment, the RNAi drug is administered in a non-buffered solution such as saline or water.

[0042] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0043] In one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a double-stranded ribonucleic acid (RNAi) agent to a subject at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0044] In another embodiment, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a double-stranded ribonucleic acid (RNAi) agent to a subject at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand being the nucleotide 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) The sequence includes a complementary region containing at least 15 consecutive nucleotides that do not exceed 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi drug contains a ligand; for example, the sense strand of the double-stranded RNAi drug is conjugated with a ligand that binds at the 3' end of the sense strand.

[0045] Double-stranded RNAi drugs can be administered to the patient in two or more doses.

[0046] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month. In another embodiment, the double-stranded RNAi drug is administered to the subject once every six weeks. In one embodiment, the double-stranded RNAi drug is administered to the subject once every two months. In yet another embodiment, the double-stranded RNAi drug is administered to the subject once a quarter.

[0047] Double-stranded RNAi drugs are, for example, approximately 0.200 to 1.825 mg / kg, 0.200 to 1.800 mg / kg, 0.200 to 1.700 mg / kg, 0.200 to 1.600 mg / kg, 0.200 to 1.500 mg / kg, 0.200 to 1.400 mg / kg, 0.200 to 1.400 mg / kg, 0.200 to 1.200 mg / kg, 0.200 to 1.100 mg / kg, 0.200 to 1.000 mg / kg, 0.200 to 0.900 mg / kg, and 0.200 to 0.800 mg / kg. g / kg, approximately 0.200 to approximately 0.700 mg / kg, approximately 0.200 to approximately 0.600 mg / kg, approximately 0.200 to approximately 0.500 mg / kg, approximately 0.200 to approximately 0.400 mg / kg, approximately 0.225 to approximately 1.825 mg / kg, approximately 0.225 to approximately 1.800 mg / kg, approximately 0.225 to approximately 1.700 mg / kg, approximately 0.225 to approximately 1.600 mg / kg, approximately 0.225 to approximately 1.500 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.200 mg / kg, approximately 0.2 25 to approximately 1,100 mg / kg, approximately 0.225 to approximately 1,000 mg / kg, approximately 0.225 to approximately 0.900 mg / kg, approximately 0.225 to approximately 0.800 mg / kg, approximately 0.225 to approximately 0.700 mg / kg, approximately 0.225 to approximately 0.600 mg / kg, approximately 0.225 to approximately 0.500 mg / kg, approximately 0.225 to approximately 0.400 mg / kg, approximately 0.250 to approximately 1.825 mg / kg, approximately 0.250 to approximately 1.800 mg / kg, approximately 0.250 to approximately 1.700 mg / kg, approximately 0.250 to approximately 1.600 mg / kg, approximately 0.250 to approximately 1.50 0 mg / kg, approximately 0.250 to approximately 1.400 mg / kg, approximately 0.250 to approximately 1.400 mg / kg, approximately 0.250 to approximately 1.200 mg / kg, approximately 0.250 to approximately 1.100 mg / kg, approximately 0.250 to approximately 1.000 mg / kg, approximately 0.250 to approximately 0.900 mg / kg, approximately 0.250 to approximately 0.800 mg / kg, approximately 0.250 to approximately 0.700 mg / kg, approximately 0.250 to approximately 0.600 mg / kg, approximately 0.250 to approximately 0.500 mg / kg, approximately 0.250 to approximately 0.400 mg / kg, approximately 0.425 to approximately 1.825 mg / kg, approximately 0.425 to approximately 1,800 mg / kg, approximately 0.425 to approximately 1,700 mg / kg, approximately 0.425 to approximately 1,600 mg / kg, approximately 0.425 to approximately 1,500 mg / kg, approximately 0.425 to approximately 1,400 mg / kg, approximately 0.425 to approximately 1,400 mg / kg, approximately 0.425 to approximately 1,200 mg / kg, approximately 0.425 to approximately 1,100 mg / kg, approximately 0.425 to approximately 1,000 mg / kg, approximately 0.425 to approximately 0.900 mg / kg, approximately 0.425 to approximately 0.800 mg / kg, approximately 0.425 to approximately 0.700 mg / kg, approximately 0.42 5 to approximately 0.600 mg / kg, approximately 0.425 to approximately 0.500 mg / kg, approximately 0.450 to approximately 1.825 mg / kg, approximately 0.450 to approximately 1.800 mg / kg, approximately 0.450 to approximately 1.700 mg / kg, approximately 0.450 to approximately 1.600 mg / kg, approximately 0.450 to approximately 1.500 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.200 mg / kg, approximately 0.450 to approximately 1.100 mg / kg, approximately 0.450 to approximately 1.000 mg / kg, approximately 0.450. From approximately 0.900 mg / kg, from approximately 0.450 to approximately 0.800 mg / kg, from approximately 0.450 to approximately 0.700 mg / kg, from approximately 0.450 to approximately 0.600 mg / kg, from approximately 0.450 to approximately 0.500 mg / kg, from approximately 0.475 to approximately 1.825 mg / kg, from approximately 0.475 to approximately 1.800 mg / kg, from approximately 0.475 to approximately 1.700 mg / kg, from approximately 0.475 to approximately 1.600 mg / kg, from approximately 0.475 to approximately 1.500 mg / kg, from approximately 0.475 to approximately 1.400 mg / kg, from approximately 0.475 to approximately 1.400 mg / kg, from approximately 0.475 to approximately 1.200 mg / kg, approximately 0.475 to approximately 1.100 mg / kg, approximately 0.475 to approximately 1.000 mg / kg, approximately 0.475 to approximately 0.900 mg / kg, approximately 0.475 to approximately 0.800 mg / kg, approximately 0.475 to approximately 0.700 mg / kg, approximately 0.475 to approximately 0.600 mg / kg, approximately 0.475 to approximately 0.500 mg / kg, approximately 0.875 to approximately 1.825 mg / kg, approximately 0.875 to approximately 1.800 mg / kg, approximately 0.875 to approximately 1.700 mg / kg, approximately 0.875 to approximately 1.600 mg / kg, approximately 0.875 to approximately 1.500 mg / kg, approximately 0. 875 to approximately 1,400 mg / kg, approximately 0.875 to approximately 1,400 mg / kg, approximately 0.875 to approximately 1,200 mg / kg, approximately 0.875 to approximately 1,100 mg / kg, approximately 0.875 to approximately 1,000 mg / kg, approximately 0.875 to approximately 0.900 mg / kg, approximately 0.900 to approximately 1.825 mg / kg, approximately 0.900 to approximately 1,800 mg / kg, approximately 0.900 to approximately 1,700 mg / kg, approximately 0.900 to approximately 1,600 mg / kg, approximately 0.900 to approximately 1,500 mg / kg, approximately 0.900 to approximately 1,400 mg / kg, approximately 0.900 to approximately 1.40 0 mg / kg, approximately 0.900 to approximately 1.200 mg / kg, approximately 0.900 to approximately 1.100 mg / kg, approximately 0.900 to approximately 1.000 mg / kg, approximately 0.925 to approximately 1.825 mg / kg, approximately 0.925 to approximately 1.800 mg / kg, approximately 0.925 to approximately 1.700 mg / kg, approximately 0.925 to approximately 1.600 mg / kg, approximately 0.925 to approximately 1.500 mg / kg, approximately 0.925 to approximately 1.400 mg / kg, approximately 0.925 to approximately 1.400 mg / kg, approximately 0.925 to approximately 1.200 mg / kg, approximately 0.925 to approximately 1.100 mg / kg, or approximately 0.It can be administered to the target population once a month at a dose of 925 to 1,000 mg / kg.

[0048] In some embodiments, the dose of the double-stranded RNAi drug is administered to the subject as a once-monthly dose of approximately 0.200 mg / kg to approximately 0.250 mg / kg; or as a once-monthly dose of approximately 0.425 mg / kg to approximately 0.475 mg / kg; or as a once-monthly dose of approximately 0.875 mg / kg to approximately 0.925 mg / kg; or as a once-monthly dose of approximately 1.775 mg / kg to approximately 1.825 mg / kg.

[0049] In one embodiment, the double-stranded RNAi drug is administered to the subject at a dose of 0.225 mg / kg once a month.

[0050] In another embodiment, the double-stranded RNAi drug is administered to the subject at a monthly dose of 0.450 mg / kg.

[0051] In yet another embodiment, the double-stranded RNAi drug is administered to the subject at a monthly dose of 0.900 mg / kg.

[0052] In one embodiment, the double-stranded RNAi drug is administered to the subject at a dose of 1,800 mg / kg once a month.

[0053] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0054] In one embodiment, administration of a double-stranded RNAi drug to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%.

[0055] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0056] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0057] In another embodiment, administration of a double-stranded RNAi drug to a subject is sufficient to achieve peak thrombin formation levels of over 40% in the subject.

[0058] In yet another embodiment, administration of a double-stranded RNAi agent to a subject at a dose thereof reduces the subject's ABR by approximately 80 to 95 percent compared to the median on-demand annual bleeding rate (ABR) of a subject who has a disorder that would benefit from reduced Serpinc1 expression and has not received the double-stranded RNAi agent.

[0059] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0060] In one embodiment, the subject is a human.

[0061] The disorder may be an acquired or hereditary bleeding disorder, such as hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0062] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0063] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0064] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0065] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0066] The complementary region may be at least 17 nucleotides or possibly 19 nucleotides long.

[0067] In one embodiment, the complementary region has a nucleotide length between 19 and 21. In another embodiment, the complementary region has a nucleotide length between 21 and 23.

[0068] In one embodiment, each chain does not exceed 30 nucleotides in length.

[0069] At least one strand of a double-stranded RNAi drug has a 3' overhang of at least one nucleotide or at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10 In some embodiments, the 3' overhang may include 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug includes a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand includes a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug include an overhang of at least 1 nucleotide.

[0070] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0071] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0072] In a particular embodiment, the ligand is as follows: [ka]

[0073] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0074] In one embodiment, X is O.

[0075] In one embodiment, the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0076] In one embodiment, the double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0077] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0078] In one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded RNAi agent to a subject, ranging from about 25 mg to about 100 mg, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 5 and at least 15 consecutive nucleotides not exceeding 3 nucleotides, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0079] In another embodiment, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded RNAi agent to a subject ranging from about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and at least 15 distinct consecutive nucleotides not exceeding 3 nucleotides. The antisense strand contains the nucleotide sequence of SEQ ID NO: 5 and at least 15 consecutive nucleotides that are not more than 3 nucleotides different, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi drug contains a ligand, for example, the sense strand of the double-stranded RNAi drug is conjugated with a ligand that binds at the 3' end of the sense strand.

[0080] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0081] In another embodiment, the sense strand and antisense strand include a complementary region containing at least 15 consecutive nucleotides that are different from any one sequence listed in either Table 2 or 3 and not exceeding 3 nucleotides.

[0082] In some embodiments, the modified nucleotide is independently selected from the group consisting of 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides bonded to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In yet another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-allyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0083] In another embodiment of the double-stranded RNAi drug, at least one strand includes a 3' overhang of at least one nucleotide. In yet another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, at least one strand of the RNAi drug includes a 5' overhang of at least one nucleotide. In a particular embodiment, at least one strand includes a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug include an overhang of at least one nucleotide.

[0084] In another embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded RNAi agent to a subject, ranging from about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding Serpinc1, and each strand being about 14 to about 30 nucleotides long, and the double-stranded RNAi agent is expressed by formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by: n p ' is a 2-nucleotide overhang, n p Each nucleotide within is linked to an adjacent nucleotide via phosphorothioate linkage; Each N a and N a' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two distinctly modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications in three consecutive nucleotides, where the modifications are 2'-O-methyl or 2'-fluorinated. It is a modifier; The sense strand and antisense strand each independently contain two phosphorothioate links at their 5' ends; The sense chain is conjugated with at least one ligand, the ligand being one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. This prevents at least one symptom in individuals with a disorder who would benefit from reduced Serpinc1 expression.

[0085] In another embodiment, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded RNAi agent to a subject ranging from about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to the mRNA encoding Serpinc1, and each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is expressed by formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by: n p ' is a 2-nucleotide overhang, n p Each nucleotide within is linked to an adjacent nucleotide via phosphorothioate linkage; Each N a and Na ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two distinctly modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications in three consecutive nucleotides, where the modifications are 2'-O-methyl or 2'-fluoro modifications; The sense strand and antisense strand each independently contain two phosphorothioate links at their 5' ends; The sense chain is conjugated with at least one ligand, the ligand being one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. This prevents at least one symptom in individuals with a disorder who would benefit from reduced Serpinc1 expression.

[0086] In one embodiment, the double-stranded RNAi drug is administered to the subject in a single dose or in two or more doses, for example, three, four, five, or six doses.

[0087] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 2 months, once a quarter, or as needed.

[0088] Double-stranded RNAi drugs are available in doses such as approximately 25 mg to 100 mg, approximately 25 mg to 95 mg, approximately 25 mg to 90 mg, approximately 25 mg to 85 mg, approximately 25 mg to 80 mg, approximately 25 mg to 75 mg, approximately 25 mg to 70 mg, approximately 25 mg to 65 mg, approximately 25 mg to 60 mg, approximately 25 mg to 50 mg, approximately 50 mg to 100 mg, and approximately 50 mg to 95 mg. Between approximately 50mg and 90mg, between approximately 50mg and 85mg, between approximately 50mg and 80mg, between approximately 30mg and 100mg, between approximately 30mg and 90mg, between approximately 30mg and 80mg, between approximately 40mg and 100mg, between approximately 40mg and 90mg, between approximately 40mg and 80mg, between approximately 60mg and 100mg, between approximately 60mg and 90mg, between approximately 25mg and 55mg, between approximately 25mg and 65mg, It can be administered to the subject as a fixed dose between approximately 30 mg and approximately 95 mg, between approximately 30 mg and approximately 85 mg, between approximately 30 mg and approximately 75 mg, between approximately 30 mg and approximately 65 mg, between approximately 30 mg and approximately 55 mg, between approximately 40 mg and approximately 95 mg, between approximately 40 mg and approximately 85 mg, between approximately 40 mg and approximately 75 mg, between approximately 40 mg and approximately 65 mg, between approximately 40 mg and approximately 55 mg, or between approximately 45 mg and approximately 95 mg, for example, as a fixed dose for 1, 2, 3, 4, 5, 6, 7, 8 months or longer.

[0089] In some embodiments, the double-stranded RNAi drug can be administered to the subject as a fixed dose of approximately 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg.

[0090] The subjects may include individuals with acquired or hereditary bleeding disorders, such as hemophilia, specifically hemophilia A, hemophilia B, or hemophilia C.

[0091] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0092] In one embodiment, administration of a double-stranded RNAi agent to a subject results in increased blood coagulation and / or decreased Serpinc1 protein accumulation.

[0093] In one embodiment, the method further includes measuring the thrombin level of the subject.

[0094] Double-stranded RNAi drugs can be administered subcutaneously or intravenously.

[0095] In one embodiment, substantially all nucleotides of the antisense strand and substantially all nucleotides of the sense strand of the RNAi drug contain modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications. In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand of the RNAi drug are modified nucleotides.

[0096] In one embodiment, the YYY motif occurs at or near the break point of the sense chain.

[0097] In one embodiment, the Y'Y'Y' motif arises at the 11th, 12th, and 13th positions from the 5' end of the antisense chain.

[0098] The double-stranded region may be 15-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-25 nucleotide pairs long, 23-27 nucleotide pairs long, 19-21 nucleotide pairs long, or 21-23 nucleotide pairs long.

[0099] Each chain may contain 15 to 30 nucleotides, or sometimes 19 to 30 nucleotides.

[0100] In one embodiment, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0101] In one embodiment, the ligand is as follows: [ka]

[0102] In one embodiment, the ligand is bound to the 3' end of the sense strand.

[0103] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0104] In one embodiment, the base pair at the first position of the 5' end of the antisense strand of the double helix is ​​an AU base pair.

[0105] In one embodiment, the RNAi agent is AD-57213 ((sense (5'→3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf (SEQ ID NO: 13); antisense (5'→3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage)).

[0106] In one embodiment, the drug is administered as a pharmaceutical composition. In another embodiment, the RNAi drug is administered in a non-buffered solution such as saline or water.

[0107] In another embodiment, siRNA is an acetate, citrate, prolamin, carbonate, or It is administered with a buffer, such as a buffer containing phosphates or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0108] In one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to a subject ranging from about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0109] In another embodiment, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to a subject, ranging from about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0110] Double-stranded RNAi drugs can be administered to the patient in two or more doses.

[0111] In some embodiments, the double-stranded RNAi drug is administered to the subject monthly, every 5 weeks, every 6 weeks, every 7 weeks, every 2 months, quarterly, or as needed.

[0112] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month. In another embodiment, the double-stranded RNAi drug is administered to the subject once every six weeks. In one embodiment, the double-stranded RNAi drug is administered to the subject once every two months. In yet another embodiment, the double-stranded RNAi drug is administered to the subject once a quarter.

[0113] Double-stranded RNAi drugs are, for example, in doses of approximately 25 mg to 100 mg, for example, between approximately 25 mg and 95 mg, between approximately 25 mg and 90 mg, between approximately 25 mg and 85 mg, between approximately 25 mg and 80 mg, between approximately 25 mg and 75 mg, between approximately 25 mg and 70 mg, between approximately 25 mg and 65 mg, between approximately 25 mg and 60 mg, between approximately 25 mg and 50 mg, between approximately 50 mg and 100 mg, between approximately 50 mg and 95 mg, between approximately 50 mg and 90 mg, between approximately 50 mg and 85 mg, between approximately 50 mg and 80 mg, between approximately 30 mg and 100 mg, between approximately 30 mg and 90 mg, and approximately 3 Between 0 mg and approximately 80 mg, between approximately 40 mg and approximately 100 mg, between approximately 40 mg and approximately 90 mg, between approximately 40 mg and approximately 80 mg, between approximately 60 mg and approximately 100 mg, between approximately 60 mg and approximately 90 mg, between approximately 25 mg and approximately 55 mg, between approximately 25 mg and approximately 65 mg, between approximately 30 mg and approximately 95 mg, between approximately 30 mg and approximately 85 mg, between approximately 30 mg and approximately 75 mg, between approximately 30 mg and approximately 65 mg, between approximately 30 mg and approximately 55 mg, between approximately 40 mg and approximately 95 mg, between approximately 40 mg and approximately 85 mg, between approximately 40 mg and approximately 75 mg, between approximately 40 mg and approximately 65 mg, between approximately 40 mg and approximately 55 mg, or from approximately 45 mg It can be administered to the target population as a fixed dose of approximately 95 mg.

[0114] In some embodiments, the double-stranded RNAi drug can be administered as a fixed dose of approximately 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg.

[0115] In some embodiments, the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 25 mg; or as a fixed dose of approximately 50 mg; or as a fixed dose of approximately 80 mg; or as a fixed dose of approximately 100 mg.

[0116] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0117] In one embodiment, administration of a double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, approximately 90% to approximately 95%, or more than 95%.

[0118] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0119] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0120] In another embodiment, administration of a double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels of approximately 40%, 45%, 50%, 55%, or more than approximately 60% in the subject.

[0121] In yet another embodiment, administration of a double-stranded RNAi agent to a subject at a dose thereof reduces the subject's median on-demand annual bleeding rate (ABR) by approximately 80% to approximately 95%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, or approximately 90% to approximately 95%, compared to the median ABR of a subject that has not received the double-stranded RNAi agent and has a disorder that would benefit from reduced Serpinc1 expression.

[0122] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0123] In one embodiment, the subject is a human.

[0124] The disorder may be an acquired or hereditary bleeding disorder, such as hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0125] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0126] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0127] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0128] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0129] The complementary region may be at least 17 nucleotides or possibly 19 nucleotides long.

[0130] In one embodiment, the complementary region has a nucleotide length between 19 and 21. In another embodiment, the complementary region has a nucleotide length between 21 and 23.

[0131] In one embodiment, each chain does not exceed 30 nucleotides in length.

[0132] In some embodiments, at least one strand of a double-stranded RNAi drug may have a 3' overhang of at least one nucleotide or a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug may have a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand may have a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug may have an overhang of at least one nucleotide.

[0133] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0134] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0135] In a particular embodiment, the ligand is as follows: [ka]

[0136] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0137] In one embodiment, X is O.

[0138] In one embodiment, the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0139] In one embodiment, the double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0140] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0141] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0142] In one embodiment, the drug is administered as a pharmaceutical composition. In another embodiment, the RNAi drug is administered in a non-buffered solution such as saline or water.

[0143] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0144] In a further embodiment, the present invention provides a kit for carrying out the method of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally means for administering the RNAi agent to a target.

[0145] In one embodiment, the present invention provides a method for preventing at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby preventing at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0146] In another aspect, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method involves a fixed dose of approximately 50 mg of double-stranded ribonucleic acid. The treatment involves administering an RNAi (RNAi) agent to a subject, with a fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand that binds at its 3' end, thereby treating a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0147] In one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby preventing at least one symptom of a disorder in a subject that would benefit from reduced Serpinc1 expression.

[0148] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby treating the subject having a disorder that would benefit from reduced Serpinc1 expression.

[0149] In one embodiment, administration of a double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, approximately 90% to approximately 95%, or more than 95%.

[0150] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0151] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0152] In another embodiment, administration of a double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels of approximately 40%, 45%, 50%, 55%, or more than approximately 60% in the subject.

[0153] In yet another embodiment, administration of a double-stranded RNAi agent to a target having impairments that would benefit from reduced Serpinc1 expression is performed. Compared to the median on-demand annual bleeding rate (ABR) of previously untreated subjects, this reduces the subject's ABR by approximately 80% to 95%, 80% to 85%, 85% to 90%, or 90% to 95%.

[0154] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0155] In one embodiment, the subject is a human.

[0156] The disorder may be an acquired bleeding disorder or a genetic bleeding disorder, such as a bleeding disorder like hemophilia, such as hemophilia A, hemophilia B, or hemophilia C.

[0157] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0158] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0159] In one embodiment, the double-stranded RNAi agent is administered to the subject long-term.

[0160] In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0161] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and unnatural bases containing nucleotides.

[0162] The complementary region may be at least 17 nucleotides long or 19 nucleotides long.

[0163] In one embodiment, the complementary region is between 19 and 21 nucleotides long. In another embodiment, the complementary region is between 21 and 23 nucleotides long.

[0164] In one embodiment, each strand does not exceed 30 nucleotides in length.

[0165] In some embodiments, at least one strand of a double-stranded RNAi drug may have a 3' overhang of at least one nucleotide or a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug may have a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand may have a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug may have an overhang of at least one nucleotide.

[0166] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand binds to the RNAi drug via a monovalent, divalent, or trivalent branched linker. It may also be one or more GalNAcs. The ligand may be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0167] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0168] In a particular embodiment, the ligand is as follows: [ka]

[0169] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0170] In one embodiment, X is O.

[0171] In one embodiment, the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0172] In one embodiment, the double-stranded RNAi drug has a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16), and 5'-UU It contains an antisense strand containing the nucleotide sequence GAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0173] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0174] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [Chemical formula] X is O or S.

[0175] In one embodiment, the agent is administered as a pharmaceutical composition. In one embodiment, an RNAi agent in a non-buffered solution such as saline or water is administered.

[0176] In another embodiment, the siRNA is administered with a buffer such as a buffer containing acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS).

[0177] In another aspect, the present invention provides a kit for performing the methods of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally, means for administering the RNAi agent to a subject.

[0178] In one aspect, the present invention provides a method of preventing at least one symptom in a subject having a disorder that would benefit from a decrease in Serpinc1 expression. The method includes administering to the subject a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent having a sense strand comprising the nucleotide sequence of 5'-G GUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugated to a ligand that binds at the 3' end, thereby preventing at least one symptom in a subject having a disorder that would benefit from a decrease in Serpinc1 expression.

[0179] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of approximately 50 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby treating the subject having a disorder that would benefit from reduced Serpinc1 expression.

[0180] In one embodiment, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0181] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby treating the subject having a disorder that would benefit from reduced Serpinc1 expression.

[0182] In one embodiment, administration of a double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, approximately 90% to approximately 95%, or more than 95%.

[0183] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0184] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0185] In another embodiment, administration of a double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels of approximately 40%, 45%, 50%, 55%, or more than approximately 60% in the subject.

[0186] In yet another embodiment, administration of a double-stranded RNAi agent to a subject at a dose thereof reduces the subject's median on-demand annual bleeding rate (ABR) by approximately 80% to approximately 95%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, or approximately 90% to approximately 95%, compared to the median ABR of a subject that has not received the double-stranded RNAi agent and has a disorder that would benefit from reduced Serpinc1 expression.

[0187] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0188] In one embodiment, the subject is a human.

[0189] The disorder may be an acquired or hereditary bleeding disorder, such as hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0190] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0191] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0192] In one embodiment, the double-stranded RNAi drug is administered to the subject over a long period of time.

[0193] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0194] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0195] In one embodiment, each chain does not exceed 30 nucleotides in length.

[0196] In some embodiments, at least one strand of a double-stranded RNAi drug may have a 3' overhang of at least one nucleotide or a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug may have a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand may have a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug may have at least one nucleotide Includes overhang.

[0197] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0198] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0199] In a particular embodiment, the ligand is as follows: [ka]

[0200] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0201] In one embodiment, X is O.

[0202] In one embodiment, the sense chain is 5'-GfsgsUfuAfaCfaCfCfAfuU The molecule contains fuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0203] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0204] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0205] In another embodiment, the present invention provides a kit for carrying out the method of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally means for administering the RNAi agent to a target.

[0206] In one embodiment, the present invention provides a method for preventing at least one symptom of hemophilia in a subject having hemophilia, e.g., hemophilia A (with or without inhibitor), hemophilia B (with or without inhibitor), or hemophilia C (with or without inhibitor). The method comprises administering a fixed dose of approximately 50 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby having a disorder that would benefit from reduced Serpinc1 expression. Prevent at least one of the target symptoms.

[0207] In another embodiment, the present invention provides a method for treating subjects having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method comprises administering a fixed dose of approximately 50 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0208] In one embodiment, the present invention provides a method for preventing at least one symptom in a person having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method involves administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) drug to a subject, the fixed dose of the double-stranded RNAi drug being administered to the subject approximately once a month, the double-stranded RNAi drug comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression.

[0209] In another embodiment, the present invention provides a method for treating subjects having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to a subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand being modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0210] In one embodiment, the present invention provides a method for preventing at least one symptom in a person having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method involves administering a fixed dose of approximately 50 mg of a double-stranded ribonucleic acid (RNAi) drug to a subject, with the fixed dose of the double-stranded RNAi drug being administered to the subject approximately once a month. The double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and an antisense strand containing the nucleotide sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; and Af, Cf, Gf, or Uf are 2'-fluoroA, C, G. t is U; s is a phosphorothioate linkage, and the sense strand conjugates with a ligand at its 3' end, thereby preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression.

[0211] In another embodiment, the present invention provides a method for treating subjects having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method comprises administering a fixed dose of approximately 50 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent having a sense strand containing the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the nucleo 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14). The antisense chain comprises a tide sequence, where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage, and the sense chain conjugates with a ligand bound at its 3' end, thereby treating subjects with impairments that would benefit from reduced Serpinc1 expression.

[0212] In one embodiment, the present invention provides a method for preventing at least one symptom of a subject having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent having a sense strand containing the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the nucleotide sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14) The antisense chain contains A, C, G, and U, where A, C, G, or U is ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage, and the sense chain conjugates with a ligand bound at its 3' terminus, thereby preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression.

[0213] In another embodiment, the present invention provides a method for treating subjects having hemophilia, for example, hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method comprises administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent being administered to the subject approximately once a month, the double-stranded RNAi agent having a sense strand containing the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the nucleo 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14). The antisense chain comprises a tide sequence, where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage, and the sense chain conjugates with a ligand bound at its 3' end, thereby treating subjects with impairments that would benefit from reduced Serpinc1 expression.

[0214] In one embodiment, administration of a double-stranded RNAi drug to a subject in a given dose results in Se in the subject. It reduces rpinc1 activity by approximately 70% to 95%, 70% to 80%, 80% to 90%, 90% to 95%, or more than 95%.

[0215] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0216] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0217] In another embodiment, administration of a double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels of approximately 40%, 45%, 50%, 55%, or more than approximately 60% in the subject.

[0218] In yet another embodiment, administration of a double-stranded RNAi agent to a subject at a dose thereof reduces the subject's median on-demand annual bleeding rate (ABR) by approximately 80% to approximately 95%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, or approximately 90% to approximately 95%, compared to the median ABR of a subject that has not received the double-stranded RNAi agent and has a disorder that would benefit from reduced Serpinc1 expression.

[0219] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0220] In one embodiment, the subject is a human.

[0221] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0222] In one embodiment, the double-stranded RNAi drug is administered to the subject over a long period of time.

[0223] In a particular embodiment, the ligand is as follows: [ka]

[0224] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0225] In one embodiment, X is O.

[0226] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0227] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0228] In another embodiment, the present invention provides a kit for carrying out the method of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally means for administering the RNAi agent to a target.

[0229] In one embodiment, the present invention provides a method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to a subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0230] In another embodiment, the present invention provides a method for treating subjects having a disorder that would benefit from reduced Serpinc1 expression. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to a subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand that binds at the 3' end of the sense strand.

[0231] Double-stranded RNAi drugs can be administered to the patient in two or more doses.

[0232] In some embodiments, the double-stranded RNAi drug is administered to the subject monthly, every 5 weeks, every 6 weeks, every 7 weeks, every 2 months, quarterly, or as needed.

[0233] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month. In another embodiment, the double-stranded RNAi drug is administered to the subject once every six weeks. In one embodiment, the double-stranded RNAi drug is administered to the subject once every two months. In yet another embodiment, the double-stranded RNAi drug is administered to the subject once a quarter.

[0234] Double-stranded RNAi drugs can be administered to patients as fixed doses, for example, between approximately 50 mg and 90 mg, between approximately 50 mg and 85 mg, between approximately 50 mg and 80 mg, between approximately 40 mg and 80 mg, between approximately 60 mg and 90 mg, between approximately 25 mg and 55 mg, between approximately 25 mg and 65 mg, between approximately 40 mg and 85 mg, between approximately 40 mg and 75 mg, between approximately 40 mg and 65 mg, or between approximately 40 mg and 55 mg.

[0235] In some embodiments, the double-stranded RNAi drug can be administered as a fixed dose of approximately 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, or 90 mg.

[0236] In some embodiments, the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 40 mg; or as a fixed dose of approximately 50 mg; or as a fixed dose of approximately 80 mg; or as a fixed dose of approximately 90 mg.

[0237] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0238] In one embodiment, administration of a double-stranded RNAi drug to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%.

[0239] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0240] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0241] In another embodiment, administration of a double-stranded RNAi drug to a subject is sufficient to achieve peak thrombin formation levels of over 40% in the subject.

[0242] In yet another embodiment, administration of a double-stranded RNAi agent to a subject at a dose thereof reduces the subject's ABR by approximately 80 to 95 percent compared to the median on-demand annual bleeding rate (ABR) of a subject who has a disorder that would benefit from reduced Serpinc1 expression and has not received the double-stranded RNAi agent.

[0243] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0244] In one embodiment, the subject is a human.

[0245] The disorder may be an acquired or hereditary bleeding disorder, such as hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0246] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0247] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0248] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0249] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0250] The complementary region may be at least 17 nucleotides or possibly 19 nucleotides long.

[0251] In one embodiment, the complementary region has a nucleotide length between 19 and 21. In another embodiment, the complementary region has a nucleotide length between 21 and 23.

[0252] In one embodiment, each chain does not exceed 30 nucleotides in length.

[0253] In some embodiments, at least one strand of a double-stranded RNAi drug may have a 3' overhang of at least one nucleotide or a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug may have a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand may have a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug may have an overhang of at least one nucleotide.

[0254] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0255] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0256] In a particular embodiment, the ligand is as follows: [ka]

[0257] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0258] In one embodiment, X is O.

[0259] In one embodiment, the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0260] In one embodiment, the double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0261] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0262] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0263] In one embodiment, the drug is administered as a pharmaceutical composition. In another embodiment, the RNAi drug is administered in a non-buffered solution such as saline or water.

[0264] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0265] In another embodiment, the present invention provides a kit for carrying out the method of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally means for administering the RNAi agent to a target.

[0266] In one embodiment, the present invention provides a method for inhibiting Serpinc1 expression in a subject. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) drug to a subject, wherein the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi drug comprises a ligand, for example, the sense strand of the double-stranded RNAi drug is conjugated with a ligand bound at the 3' end of the sense strand.

[0267] In another embodiment, the present invention provides a method for inhibiting Serpinc1 expression in a subject. The method comprises administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to a subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand bound at the 3' end of the sense strand.

[0268] Double-stranded RNAi drugs can be administered to the patient in two or more doses.

[0269] In some embodiments, the double-stranded RNAi drug is administered to the subject once a month, once every 5 weeks, and 6 It is administered once a week, once every 7 weeks, once every 2 months, once a quarter, or as needed.

[0270] In one embodiment, the double-stranded RNAi drug is administered to the subject once a month. In another embodiment, the double-stranded RNAi drug is administered to the subject once every six weeks. In one embodiment, the double-stranded RNAi drug is administered to the subject once every two months. In yet another embodiment, the double-stranded RNAi drug is administered to the subject once a quarter.

[0271] Double-stranded RNAi drugs can be administered to patients as fixed doses, for example, between approximately 50 mg and 90 mg, between approximately 50 mg and 85 mg, between approximately 50 mg and 80 mg, between approximately 40 mg and 80 mg, between approximately 60 mg and 90 mg, between approximately 25 mg and 55 mg, between approximately 25 mg and 65 mg, between approximately 40 mg and 85 mg, between approximately 40 mg and 75 mg, between approximately 40 mg and 65 mg, or between approximately 40 mg and 55 mg.

[0272] In some embodiments, the double-stranded RNAi drug can be administered as a fixed dose of approximately 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, or 90 mg.

[0273] In some embodiments, the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 40 mg; or as a fixed dose of approximately 50 mg; or as a fixed dose of approximately 80 mg; or as a fixed dose of approximately 90 mg.

[0274] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0275] In one embodiment, administration of a double-stranded RNAi drug to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%.

[0276] In one embodiment, the subject is a human.

[0277] In one embodiment, the subject has a disorder that would benefit from reduced Serpinc1 expression. The disorder may be an acquired or hereditary bleeding disorder, such as hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0278] In one embodiment, the subject has hemophilia A and is an inhibitor target. In another embodiment, the subject has hemophilia B and is an inhibitor target. In yet another embodiment, the subject has hemophilia C and is an inhibitor target.

[0279] In another embodiment, administration of a double-stranded RNAi drug to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a subject without impairment that would benefit from reduced Serpinc1 expression.

[0280] In one embodiment, the administration of a dose of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administering factor VIII to the subject.

[0281] In another embodiment, administration of a double-stranded RNAi drug to a subject is sufficient to achieve peak thrombin formation levels of over 40% in the subject.

[0282] In yet another embodiment, the administration of a double-stranded RNAi drug to a target is performed by Serp This treatment reduces the median on-demand annual bleeding rate (ABR) of subjects with disorders that would benefit from reduced inc1 expression, by approximately 80 to 95%, compared to their historical median ABR of subjects not receiving double-stranded RNAi agents.

[0283] In one embodiment, the double-stranded RNAi drug is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0284] In one embodiment, the double-stranded RNAi drug is administered subcutaneously to the subject.

[0285] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0286] In one embodiment, the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0287] The complementary region may be at least 17 nucleotides or possibly 19 nucleotides long.

[0288] In one embodiment, the complementary region has a nucleotide length between 19 and 21. In another embodiment, the complementary region has a nucleotide length between 21 and 23.

[0289] In one embodiment, each chain does not exceed 30 nucleotides in length.

[0290] In some embodiments, at least one strand of a double-stranded RNAi drug may have a 3' overhang of at least one nucleotide or a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi drug may have a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand may have a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi drug may have an overhang of at least one nucleotide.

[0291] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may also be one or more GalNAcs conjugated to the RNAi drug via a monovalent, divalent, or trivalent branched linker. The ligand may also be conjugated to the 3' end of the sense strand of the double-stranded RNAi drug, the 5' end of the sense strand of the double-stranded RNAi drug, the 3' end of the antisense strand of the double-stranded RNAi drug, or the 5' end of the antisense strand of the double-stranded RNAi drug.

[0292] In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0293] In a particular embodiment, the ligand is as follows: [ka]

[0294] In one embodiment, the RNAi drug is conjugated with a ligand as shown in the schematic diagram below, [ka] X is either O or S.

[0295] In one embodiment, X is O.

[0296] In one embodiment, the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0297] In one embodiment, the double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0298] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage.

[0299] In one embodiment, the sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage; and the sense chain is conjugated with a ligand as shown in the schematic diagram below. [ka] X is either O or S.

[0300] In one embodiment, the drug is administered as a pharmaceutical composition. In another embodiment, the RNAi drug is administered in a non-buffered solution such as saline or water.

[0301] In another embodiment, siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0302] In another embodiment, the present invention provides a kit for carrying out the method of the present invention. The kit may include the RNAi agent of the present invention, instructions for use, and optionally means for administering the RNAi agent to a target. [Brief explanation of the drawing]

[0303] [Figure 1A] Figure 1A is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma thrombin formation levels in one healthy human subject. [Figure 1B] Figure 1B is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma thrombin formation levels in one healthy human subject. [Figure 1C] Figure 1C is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma thrombin formation levels in one healthy human subject. [Figure 1D] Figure 1D is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma thrombin formation levels in one healthy human subject. [Figure 2A]Figure 2A is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma AT(Serpinc1) protein levels in one healthy human subject. [Figure 2B] Figure 2B is a graph showing the effect of a single subcutaneous administration of 0.03 mg / kg of AD-57213 on plasma AT(Serpinc1) protein levels in one healthy human subject. [Figure 3] Figure 3 is a graph showing the relationship between the rate of AT(Serpinc1) knockdown and the rate of increased peak thrombin formation in healthy subjects who received a single subcutaneous dose of 0.03 mg / kg of AD-57213. [Figure 4] Figure 4 is a graph showing the effects of multiple doses of AD-57213 at 0.015 mg / kg, 0.045 mg / kg, or 0.075 mg / kg on plasma AT(Serpinc1) protein levels in human subjects with hemophilia A or B. [Figure 5A] Figure 5A is a graph showing the effects of multiple doses of AD-57213 at 0.225 mg / kg, 0.450 mg / kg, 0.900 mg / kg, 1.800 mg / kg, or 80 mg on plasma AT(Serpinc1) protein levels in human subjects with hemophilia A or B. [Figure 5B] Figure 5B is a graph showing the dose-dependent effect of AD-57213 on plasma AT(Serpinc1) protein levels in human subjects. [Figure 6A] Figure 6A is a graph showing the effect of multiple doses of AD-57213 at 0.015 mg / kg or 0.045 mg / kg on peak thrombin levels in human subjects with hemophilia A or B. [Figure 6B] Figure 6B is a graph showing the effect of multiple doses of AD-57213 at 0.015 mg / kg or 0.045 mg / kg on thrombin formation in human subjects with hemophilia A or B, expressed as a percentage change compared to the baseline group. [Figure 7]Figure 7 is a graph showing the effect of multiple doses of 0.045 mg / kg AD-57213 on clot formation time and clotting time in one subject with hemophilia A (subjects 101-009). [Figure 8] Figure 8 is a graph showing the average maximum AT reduction with an equivalent dose administered once a month. [Figure 9] Figure 9 is a graph showing the effect of multiple doses of AD-57213 on thrombin formation due to AT quartile reduction. [Figure 10A] Figure 10A is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in subjects administered 225 mcg / kg qM of AD-57213. [Figure 10B] Figure 10B is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in subjects administered 1800 mcg / kg qM of AD-57213. [Figure 10C] Figure 10C is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, as measured in subjects administered 80 mg qM of AD-57213. [Figure 11] Figure 11 is a graph showing the effect of multiple doses of AD-57213 on bleeding events due to AT quartile reduction. [Figure 12] Figure 12 is a table showing bleeding event data for subjects enrolled in Part C of the Phase I clinical trial of AD-57213. [Figure 13A] Figure 13A is a graph showing the median annual bleeding rate (ABR) before the start of the trial, at the start of the trial, and during the observation portion of the trial for all dosing cohorts in Part C of the Phase I clinical trial of AD-57213. [Figure 13B] Figure 13B is a graph showing the median annual bleeding rate (ABR) before the start of the trial, at the start of the trial, and during the observational portion of the trial for the monthly 80 mg (80 mg qM × 3) cohort in Part C of the Phase I clinical trial of AD-57213. [Figure 14A] Figure 14A is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects who received a fixed dose of 50 mg of AD-57213 once a month. [Figure 14B] Figure 14B is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects who received a fixed dose of 50 mg of AD-57213 once a month. [Figure 14C] Figure 14C is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects administered a fixed dose of 50 mg of AD-57213 once a month. [Figure 14D] Figure 14D is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects administered a fixed dose of 50 mg of AD-57213 once a month. [Figure 14E] Figure 14E is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects administered a fixed dose of 50 mg of AD-57213 once a month. [Figure 14F] Figure 14F is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects who received a fixed dose of 50 mg of AD-57213 once a month. [Figure 15] Figure 15 is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 on mean AT(Serpinc1) activity at baseline in human subjects with hemophilia A or B who have inhibitors. [Figure 16] Figure 16 is a graph showing that the AT-lowering effect of multiple doses of 50 mg AD-57213 correlates with increased thrombin formation in subjects with hemophilia A. [Figure 17A]Figure 17A is a table showing bleeding event data for subjects enrolled in Part D of the Phase I clinical trial of AD-57213. [Figure 17B] Figure 17B is a graph showing the median annual bleeding rate (ABR) before the start of the trial, at the start of the trial, and during the observation portion of the trial for all subjects in Part D of the Phase I clinical trial of AD-57213. [Figure 18] Figure 18 is a graph showing the effect of multiple doses of 80 mg of AD-57213 on mean AT(Serpinc1) activity relative to baseline in human subjects with hemophilia without inhibitors in a Phase II Open Label Extension (OLE) trial of AD-57213. [Figure 19A] Figure 19A is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 on mean AT(Serpinc1) activity relative to baseline in human subjects with hemophilia A or B without inhibitors in a Phase II Open Label Extension (OLE) trial of AD-57213. [Figure 19B] Figure 19B is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 on peak thrombin formation in human subjects with hemophilia A or B without inhibitors in a Phase II Open-Label Extension (OLE) study of AD-57213. The shaded portion of the graph represents the range of peak thrombin levels observed in healthy human volunteers (HVs) administered AD-57213, indicating less than 25% AT knockdown in the Phase I study of AD-57213 described in Example 1. The dotted line within the HV range represents the median peak thrombin level observed in healthy human volunteers (HVs), indicating less than 25% AT knockdown due to AD-57213 administration in the Phase I study of AD-57213 described in Example 1. [Figure 20A] Figure 20A is a table showing bleeding event data for subjects enrolled in the Phase II OLE clinical trial of AD-57213. [Figure 20B]Figure 20B is a graph showing the median annual bleeding rate (ABR) for all subjects in the Phase II OLE clinical trial of AD-57213, before the start of the trial, at the start of the trial, and during the observational portion of the trial. [Modes for carrying out the invention]

[0304] This invention is at least in part based on the remarkable discovery that very low doses (e.g., doses at least about 30 times lower than those taught in the art) of GalNAc-binding double-stranded RNAi agents containing specific chemical modifications exhibit extraordinary potency in inhibiting Serpinc1 expression, as well as an extraordinary duration of inhibition of Serpinc1 expression. Specifically, low-dose RNAi agents containing a GalNAc ligand, where substantially all nucleotides are modified nucleotides, such as one or more motifs of three identical modifications in three consecutive nucleotides, six phosphorothioate linkages, and a GalNAc ligand, have been shown herein to be exceptionally effective and long-lasting in silencing the activity of the Serpinc1 gene.

[0305] Accordingly, the present invention provides a method for preventing bleeding in subjects having a bleeding disorder, such as hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), which would benefit from inhibiting or reducing the expression of the Serpinc1 gene, using an iRNA composition that induces RNA-induced silencing complex (RISC)-related cleavage of the RNA transcript of the Serpinc1 gene. The present invention further provides a method for treating subjects having a bleeding disorder, such as hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), which would benefit from inhibiting or reducing the expression of the Serpinc1 gene, using an iRNA composition that induces RNA-induced silencing complex (RISC)-related cleavage of the RNA transcript of the Serpinc1 gene.

[0306] iRNA agents for use in the method of the present invention are generally about 30 nucleotides or less in length, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, The region includes an RNA strand (antisense strand) having a region of 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, and this region is substantially complementary to at least a portion of the mRNA transcript of the Serpinc1 gene.

[0307] In other embodiments, one or both strands of the double-stranded RNAi drug of the present invention are up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long, and have a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the Serpinc1 gene. In some embodiments, the sense strand and antisense strand form a double helix of 18-30 consecutive nucleotides.

[0308] In some embodiments, the iRNA agent for use in the method of the present invention can be up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long, and comprises an RNA chain (antisense chain) having a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the Serpinc1 gene. In some embodiments, such an iRNA agent having a longer antisense chain may also include a second RNA chain (sense chain) of 20-60 nucleotides long, and the sense and antisense chains form a double helix of 18-30 consecutive nucleotides.

[0309] The following detailed description concerns the preparation and use of compositions containing iRNA for inhibiting the expression of the Serpinc1 gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders who would benefit from the inhibition and / or reduction of this gene's expression. I will disclose the method to Rabi.

[0310] I. Definition To facilitate understanding of the present invention, certain terms are first defined. Furthermore, whenever parameter values ​​or ranges of values ​​are mentioned, it should be noted that intermediate values ​​and ranges of those values ​​are also intended to be part of the present invention.

[0311] The articles “a and an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements, e.g., multiple elements.

[0312] The term “including” is used herein to mean “including, but not limited to,” and is used synonymously with it.

[0313] The term "or" is used herein to mean and is used synonymously with the term "and / or" unless the context clearly indicates otherwise.

[0314] As used herein, “Serpinc1” refers to a specific polypeptide expressed in cells. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin; AT), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of human Serpinc1 mRNA can be found, for example, in GenBank accession number GI:254588059 (NM_000488; SEQ ID NO: 1). The sequence of rhesus monkey Serpinc1 mRNA can be found, for example, in GenBank accession number GI:157167169 (NM_001104583; SEQ ID NO: 2). The sequence of mouse Serpinc1 mRNA can be found, for example, in GenBank accession number GI:237874216 (NM_080844; SEQ ID NO: 3). The sequence of rat Serpinc1 mRNA can be found, for example, in GenBank accession number GI:58865629 (NM_001012027; Sequence ID No. 4).

[0315] The term “Serpinc1” also, as used herein, refers to specific polypeptides expressed in cells due to naturally occurring DNA sequence diversity in the Serpinc1 gene, such as single nucleotide polymorphisms (SNPs) in the Serpinc1 gene. Numerous SNPs within the Serpinc1 gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp). Non-exclusive examples of SNPs within the Serpinc1 gene can be found in NCBI dbSNP accessions rs677;rs5877;rs5878;rs5879;rs941988;rs941989;rs1799876;rs19637711;rs2008946; and rs2227586.

[0316] As used herein, “Subject” refers to mammals, including primates (humans, non-human primates, e.g., monkeys, and chimpanzees), non-primates (cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, and whales), or animals such as birds (e.g., ducks or geese). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced Serpinc1 expression as described herein; a human being at risk of a disease, disorder or condition that would benefit from reduced Serpinc1 expression; a human being having a disease, disorder or condition that would benefit from reduced Serpinc1 expression; and / or a disease, disorder that would benefit from reduced Serpinc1 expression. Alternatively, it could be a person who is being treated for a condition.

[0317] As used herein, the terms “to treat” or “treatment” refer to a beneficial or desired outcome, including but not limited to the alleviation or improvement of one or more symptoms, whether detectable or undetectable; a reduction in the degree of bleeding; stabilization of bleeding (i.e., no worsening); or improvement or reduction of bleeding. “Treatment” may also mean an extension of survival compared to the survival expected without treatment. In the methods of the present invention, treatment includes on-demand treatment and control, perioperative management of bleeding, and usual prophylaxis to reduce the frequency of bleeding symptoms.

[0318] The term “reduce” in relation to the level of Serpinc1 or a marker or symptom of the disease in a subject refers to a statistically significant reduction in such a level. The reduction may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, and is preferably reduced to a level that is acceptable and within the normal range for individuals without such disorder.

[0319] As used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder, or condition that would benefit from reduced expression of the Serpinc1 gene, a reduction in the likelihood of an individual developing a disease, disorder, or condition, such as symptoms associated with the disease, disorder, or condition, including bleeding. For example, if an individual with one or more risk factors for bleeding has the same risk factors but does not develop bleeding, or develops bleeding but with less severity, then the likelihood of bleeding is reduced. Effective prevention is considered to be the absence of the disease, disorder, or condition, or a reduction in the development of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically recognized scale for the disease or disorder), or a delay in symptoms (e.g., by days, weeks, months, or years).

[0320] As used herein, the term “hemorrhagic disorder” means a disease or disorder resulting in poor blood clotting and / or excessive bleeding. A hemorrhagic disorder may be a genetic disorder such as hemophilia or von Willebrand disease, or an acquired disorder associated with, for example, disseminated intravascular coagulation, pregnancy-related eclampsia, vitamin K deficiency, autoimmune disorders, inflammatory bowel disease, ulcerative colitis, skin disorders (e.g., psoriasis, pemphigus), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease), allergic drug reactions, for example, as a result of drugs such as aspirin, heparin, and warfarin, diabetes mellitus, acute hepatitis B infection, acute hepatitis C infection, malignant tumors or solid tumors (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney, and / or hematological malignancies). In one embodiment, the genetic hemorrhagic disorder is hemophilia, for example, hemophilia A, B, or C. In one embodiment, a subject having a hereditary bleeding disorder, such as hemophilia, is equipped with an inhibitor to replacement therapy, such as an alloantibody inhibitor, and is referred to herein as the "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.

[0321] "Therapeutic dose," as used herein, is the amount of RNAi agent administered to a subject with bleeding disorders and bleeding that, when administered, results in treatment of the disease (for example, by reducing, improving, or maintaining the symptoms of the pre-existing disease or one or more symptoms of the disease). The amount is intended to include. The “therapeutic effective dose” may vary depending on the RNAi drug, how the drug is administered, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, type of preceding or concomitant treatment, and any other individual characteristics of the person being treated.

[0322] Where used herein, “prophylactic effective dose” is intended to contain an amount of iRNA sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with a bleeding disorder but not bleeding, for example, a subject with a bleeding disorder and for whom surgery is planned (e.g., perioperative treatment). Improving the disease includes delaying the course of the disease or reducing the severity of the disease if it develops later. The “prophylactic effective dose” may vary depending on the iRNA, how the drug is administered, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, type of preceding or concomitant treatment, and any other individual characteristics.

[0323] The “therapeutic effective dose” or “preventive effective dose” also includes the amount of RNAi agent that produces some desired local or systemic effect with a reasonable benefit / risk ratio for any treatment. The iRNA used in the method of the present invention can be administered in an amount sufficient to produce a suitable reasonable benefit / risk ratio for such treatment.

[0324] The term "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues within the bounds of appropriate medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or impairments, corresponding to a reasonable benefit / risk ratio.

[0325] When used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc, or stearic acid), or a solvent that surrounds a material involved in the transport or delivery of the compound of interest from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, e.g., propylene glycol (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) Physiological saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffers; (21) Polyesters, polycarbonates, and / or polyacid anhydrides; (22) Expanders, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; and (22) Other non-toxic and suitable substances used in pharmaceutical formulations.

[0326] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Serpinc1 gene, and is the primary transcript product. The sequence contains mRNA, which is a product of RNA processing. In one embodiment, the target portion of the sequence is long enough to act as a substrate for iRNA-induced cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the Serpinc1 gene.

[0327] The target sequence is approximately 9-36 nucleotides long, for example, it may be approximately 15-30 nucleotides long. For example, the target sequence is approximately 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19 Possible nucleotide lengths are ~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, or 21~22 nucleotides. Intermediate ranges and lengths beyond those listed above are also intended to be part of the present invention.

[0328] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides indicated by a sequence referred to using standard nucleotide nomenclature.

[0329] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, naturally, the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or alternative substitution sites (see, for example, Table 1), which are further detailed below. Those skilled in the art will know that guanine, cytosine, adenine, and uracil can be substituted for other sites without substantially altering the base-pairing properties of oligonucleotides containing such substitution sites. For example, without limitation, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine in the nucleotide sequences of dsRNAs addressed in this invention can be substituted, for example, with nucleotides containing inosine. In another example, any adenine and cytosine in an oligonucleotide may be substituted with guanine and uracil to form GU fluctuation base pairs with target mRNA, respectively. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0330] In this specification, the terms “iRNA,” “RNAi agent,” “iRNA drug,” and “RNA interference agent,” used synonymously, refer to agents containing RNA that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as defined herein. iRNAs induce sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs modulate, for example, inhibit the expression of Serpinc1 in cells within a subject, such as a mammalian subject.

[0331] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a Serpinc1 target mRNA sequence, to induce cleavage of the target RNA. While we do not wish to be bound by theory, it is hypothesized that a long double-stranded RNA introduced into a cell is degraded into siRNA by a type III endonuclease known as Dicer. (Sharp et al., Genes Dev. 15, 2001: p. 485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein et al., Nature 409, 2001: p. 363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to lead to target recognition (Nykanen et al., Cell 107, 2001: p. 309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir et al., Genes Dev. 15, 2001: p. 188). Accordingly, in one embodiment, the present invention relates to single-stranded RNA (siRNA) formed in a cell, which promotes the formation of a RISC complex, thereby causing silencing of a target gene, namely the Serpinc1 gene. Accordingly, the term "siRNA" is used herein to also refer to RNAi as described above.

[0332] In another embodiment, the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease, Argonaut 2, which then cleaves the target mRNA. Single-stranded siRNAs are typically 15–30 nucleotides long and are chemically modified. For the design and testing of single-stranded siRNAs, see U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or as single-stranded siRNAs chemically modified by the methods described herein by Lima et al., (2012) Cell 150:883–894.

[0333] In another embodiment, the “iRNA” for use in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred herein as “double-stranded RNAi drug,” “double-stranded RNA (dsRNA) molecule,” “dsRNA drug,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands referred to as having “sense” and “antisense” orientations with respect to the Serpinc1 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein as RNA interference or RNAi.

[0334] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, “RNAi agents” may also contain chemically modified ribonucleotides; RNAi agents may also contain significant modifications to multiple nucleotides.

[0335] As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleoside linkage, and / or a modified nucleic acid base. Therefore, the term “modified nucleotide” encompasses, for example, the substitution, addition, or removal of a functional group or atom to the nucleoside linkage, sugar moiety, or nucleic acid base. Suitable modifications for use in the agents of the present invention include any type of modification disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification used in an siRNA-type molecule is referred to as “RN It is included in "Animal Pharmaceuticals".

[0336] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA by the RISC pathway, ranging from approximately 9 to 36 base pairs in length, for example, approximately 15 to 30 base pairs, for example, approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 2 The lengths may also be in the range of 0-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between those listed above are also intended to be part of this invention.

[0337] The two strands forming a double helix structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are parts of a larger molecule, and thus linked by a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, this linking RNA chain is referred to as a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

[0338] When two substantially complementary strands of dsRNA are contained by separate RNA molecules, these molecules do not need to be covalently bonded, but they can be. If the two strands are covalently bonded by something other than a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, forming a double-stranded structure, the linking structure is called a "linker." RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double helix. In addition to the double-stranded structure, RNAi may also contain one or more nucleotide overhangs.

[0339] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, e.g., a Serpinc1 target mRNA sequence, to induce cleavage of the target RNA. While we do not wish to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein et al., (2001) Nature 409:363). siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to lead to target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir et al., (2001) )Genes Dev.15:188).

[0340] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, e.g., a dsRNA. For example, if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, a nucleotide overhang exists, and vice versa. A dsRNA may contain an overhang of at least one nucleotide; or an overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. An overhang may be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of an overhang may be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0341] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0342] In certain embodiments, overhangs on the sense strand, antisense strand, or both strands may include extended lengths longer than 10 nucleotides, for example, 10–30 nucleotides, 10–25 nucleotides, 10–20 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located on the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located on the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides in the extended overhang are substituted with a nucleoside thiophosphate.

[0343] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the ends of a double-stranded RNAi drug, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi drug is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at any end of the molecule. Examples of RNAi drugs of the present invention include RNAi drugs having a nucleotide overhang at one end (i.e., a drug having one overhang and one blunt end) or RNAi drugs having nucleotide overhangs at both ends.

[0344] The terms “antisense strand” or “guide strand” refer to a strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., Serpinc1 mRNA. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., the target sequence, e.g., the Serpinc1 nucleotide sequence as defined herein. In some cases, mismatches may exist within the molecule's internal or terminal regions. Generally, the most acceptable mismatches are located within terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of an iRNA.

[0345] When used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand region as defined herein.

[0346] As used herein, the term “cleavage region” refers to a region located immediately adjacent to a cleavage site. A cleavage site is the target site where cleavage occurs. In some embodiments, the cleavage region includes one end of the cleavage site and three bases immediately adjacent to it. In some embodiments, the cleavage region includes one end of the cleavage site and two bases immediately adjacent to it. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand are bound, and the cleavage region includes nucleotides 11, 12, and 13.

[0347] Where used herein, unless otherwise indicated, the term “complementary” when used to describe a first nucleotide sequence with respect to a second nucleotide sequence means, as those skilled in the art will understand, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure. Such conditions may be stringent conditions, for example, which may include: washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook et al., (1989) Cold Spring Harbor Laboratory Press) followed by 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, and 12–16 hours at 50°C or 70°C). Other conditions may apply, such as physiologically relevant conditions that may be encountered within living organisms. Those skilled in the art can determine the set of conditions most appropriate for testing the complementarity of the two sequences, according to the final use of the hybridized nucleotides.

[0348] Complementary sequences within iRNA, for example, within dsRNA as described herein, include base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence over the full length of one or both nucleotide sequences with an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary” to each other. However, where the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary or may form one or more, but generally at most 5, 4, 3, or 2, mismatched base pairs during hybridization of a double helix of up to 30 base pairs, while retaining their ability to hybridize under conditions most relevant to their end use, e.g., inhibition of gene expression by the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, where the longer oligonucleotide has a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be referred to as "perfectly complementary" for the purposes described herein.

[0349] As used herein, “complementary” sequences refer to their ability to hybridize. Insofar as the above requirements are met, base pairs formed from non-Watson-Crick base pairs and / or non-natural and modified nucleotides may also be included, or may be formed entirely from them. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen base pairings.

[0350] The terms “complementary,” “fully complementary,” and “substantially complementary” can be used herein to describe base matches between the sense and antisense strands of a dsRNA, or between the antisense strand and target sequence of an iRNA drug, as will be understood from the context of their use.

[0351] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA in question (e.g., the mRNA encoding Serpinc1). For example, a polynucleotide is complementary to at least a portion of Serpinc1 mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding Serpinc1.

[0352] Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target Serpinc1 sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target Serpinc1 sequence and include a contiguous nucleotide sequence that is at least about 80% complementary, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over the corresponding region of the nucleotide sequence of SEQ ID NO: 1, or over the fragment of SEQ ID NO: 1 and its entire length.

[0353] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, and consequently complementary to the target Serpinc1 sequence, wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over the corresponding region of the nucleotide sequence of SEQ ID NO: 5, or any one fragment of SEQ ID NO: 5 and its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0354] In one aspect of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits target mRNA by an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by forming base pairs with mRNA and physically interfering with the translation mechanism. See Dias, N. et al., (2002) Mol Cancer Ther 1: pp. 347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides long and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0355] As used herein, the term “inhibit” is synonymous with “reduce,” “silence,” “downcontrol,” “suppress,” and other similar terms, and includes all levels of inhibition.

[0356] When used herein, the phrase "inhibiting Serpinc1 expression" includes inhibiting the expression of any Serpinc1 gene encoding the Serpinc1 protein (e.g., mouse Serpinc1 gene, rat Serpinc1 gene, monkey Serpinc1 gene, or human Serpinc1 gene) as well as Serpinc1 gene variants or mutants.

[0357] "Inhibiting the expression of the Serpinc1 gene" includes any level of inhibition of the Serpinc1 gene, such as inhibition of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%, e.g., at least partial suppression of the expression of the Serpinc1 gene.

[0358] Serpinc1 gene expression can be assessed based on the levels of any variable associated with Serpinc1 gene expression, such as Serpinc1 mRNA levels, Serpinc1 protein levels, or, for example, thrombin:antithrombin complex levels as a measure of thrombin formation ability, bleeding time, prothrombin time (PT), platelet count, and / or activated partial thromboplastin time (aPTT). Inhibition can be assessed by a decrease in the absolute or relative levels of one or more of these variables compared to control levels. Control levels may be any type of control level available in the art, such as baseline levels before administration, or levels obtained from similar subjects, cells, or samples that are untreated or treated with a control (e.g., a buffer-only control or an inactive drug control).

[0359] In one embodiment, at least partial suppression of Serpinc1 gene expression is evaluated by a decrease in the amount of Serpinc1 mRNA that can be isolated from or detected from the first cells or cell population treated to inhibit Serpinc1 gene expression, compared to a second cell population (control cells) that is substantially identical to the first cell population but not treated to inhibit Serpinc1 gene expression. The degree of inhibition can be expressed as follows:

number

[0360] The phrase "contacting an RNAi drug with cells," such as dsRNA, as used herein, includes contacting cells by any possible means. Contacting an RNAi drug with cells includes contacting an iRNA with cells in vitro or in vivo. Contact can be carried out directly or indirectly. Thus, for example, an RNAi drug can be physically brought into contact with cells by individual executions of the method, or an RNAi drug can be brought into contact with cells, or the situation can be such that it subsequently comes into contact with cells.

[0361] In vitro cell contact can be performed, for example, by incubating cells with RNAi drugs. In vivo cell contact can be performed, for example, by incubating cells This can be done by injecting the RNAi drug into or near a tissue, or by injecting the RNAi drug into another region, such as the bloodstream or subcutaneous space, so that the RNAi drug reaches the tissue to which the cells to be contacted are located. For example, the RNAi drug may contain and / or be bound to a ligand that induces the RNAi drug to the target site, such as the liver. A combination of in vitro and in vivo contact methods is also possible. For example, cells may be contacted with the RNAi drug in vitro and then transplanted into the target.

[0362] In one embodiment, contacting iRNA with cells includes “introducing” or “delivering iRNA to cells” by promoting or causing uptake or absorption into cells. Absorption or uptake of iRNA can be carried out by unassisted diffusion processes or active intracellular processes, or by adjuvants or devices. Introducing iRNA into cells is possible in vitro and / or in vivo. For example, with respect to in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be carried out by beta-glucan delivery systems such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Publication No. 2005 / 0281781, whose entire contents are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below herein and / or known in the art.

[0363] II. Method of the present invention The present invention provides therapeutic and prophylactic methods comprising administering an iRNA agent or a pharmaceutical composition comprising the iRNA agent of the present invention to a subject having a Serpinc1-related disease, such as a bleeding disorder, such as hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C). In some embodiments of the present invention, the method further comprises administering an additional therapeutic agent to the subject.

[0364] In certain embodiments of the present invention, for example, if the double-stranded RNAi drug comprises one or more motifs of three identical modifications on three consecutive nucleotides, including one motif at or near the drug cleavage site, six phosphorothioate linkages, and a GalNAc ligand, then such a drug may be administered in doses of approximately 0.200 to 1.825 mg / kg, 0.200 to approximately 1.800 mg / kg, approximately 0.200 to approximately 1.700 mg / kg, and approximately 0.200 to approximately 1.60 mg / kg. 0 mg / kg, approximately 0.200 to approximately 1.500 mg / kg, approximately 0.200 to approximately 1.400 mg / kg, approximately 0.200 to approximately 1.400 mg / kg, approximately 0.200 to approximately 1.200 mg / kg, approximately 0.200 to approximately 1.100 mg / kg, approximately 0.200 to approximately 1.000 mg / kg, approximately 0.200 to approximately 0.900 mg / kg, approximately 0.200 to approximately 0.800 mg / kg, approximately 0.200 to approximately 0.700 mg / kg, approximately 0.200 to approximately 0.600 mg / kg, approximately 0. 200 to approximately 0.500 mg / kg, approximately 0.200 to approximately 0.400 mg / kg, approximately 0.225 to approximately 1.825 mg / kg, approximately 0.225 to approximately 1.800 mg / kg, approximately 0.225 to approximately 1.700 mg / kg, approximately 0.225 to approximately 1.600 mg / kg, approximately 0.225 to approximately 1.500 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.400 mg / kg, approximately 0.225 to approximately 1.200 mg / kg, approximately 0.225 to approximately 1.10 0 mg / kg, approximately 0.225 to approximately 1.000 mg / kg, approximately 0.225 to approximately 0.900 mg / kg, approximately 0.225 to approximately 0.800 mg / kg, approximately 0.225 to approximately 0.700 mg / kg, approximately 0.225 to approximately 0.600 mg / kg, approximately 0.225 to approximately 0.500 mg / kg, approximately 0.225 to approximately 0.400 mg / kg, approximately 0.250 to approximately 1.825 mg / kg, approximately 0.250 to approximately 1.800 mg / kg, approximately 0.250 to approximately 1.700 mg / kg, approximately 0. 250 to approximately 1,600 mg / kg, approximately 0.250 to approximately 1,500 mg / kg, approximately 0.250 to approximately 1,400 mg / kg, approximately 0.250 to approximately 1,400 mg / kg, approximately 0.250 to approximately 1,200 mg / kg, approximately 0.250 to approximately 1,100 mg / kg, approximately 0.250 to approximately 1,000 mg / kg, approximately 0.250 to approximately 0.900 mg / kg, approximately 0.250 to approximately 0.800 mg / kg, approximately 0.250 to approximately 0.700 mg / kg, approximately 0.250 to approximately 0.600 mg / kg, approximately 0.250 to approximately 0.500 mg / kg, approximately 0.250 to approximately 0.40 0 mg / kg, approximately 0.425 to approximately 1.825 mg / kg, approximately 0.425 to approximately 1.800 mg / kg, approximately 0.425 to approximately 1.700 mg / kg, approximately 0.425 to approximately 1.600 mg / kg, approximately 0.425 to approximately 1.500 mg / kg, approximately 0.425 to approximately 1.400 mg / kg, approximately 0.425 to approximately 1.400 mg / kg, approximately 0.425 to approximately 1.200 mg / kg, approximately 0.425 to approximately 1.100 mg / kg, approximately 0.425 to approximately 1.000 mg / kg, approximately 0.425 to approximately 0.900 mg / kg, approximately 0.425 to approximately 0.800 mg / kg, approximately 0. 425 to approximately 0.700 mg / kg, approximately 0.425 to approximately 0.600 mg / kg, approximately 0.425 to approximately 0.500 mg / kg, approximately 0.450 to approximately 1.825 mg / kg, approximately 0.450 to approximately 1.800 mg / kg, approximately 0.450 to approximately 1.700 mg / kg, approximately 0.450 to approximately 1.600 mg / kg, approximately 0.450 to approximately 1.500 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.400 mg / kg, approximately 0.450 to approximately 1.200 mg / kg, approximately 0.450 to approximately 1.100 mg / kg, approximately 0.450 to approximately 1.00 0 mg / kg, approximately 0.450 to approximately 0.900 mg / kg, approximately 0.450 to approximately 0.800 mg / kg, approximately 0.450 to approximately 0.700 mg / kg, approximately 0.450 to approximately 0.600 mg / kg, approximately 0.450 to approximately 0.500 mg / kg, approximately 0.475 to approximately 1.825 mg / kg, approximately 0.475 to approximately 1.800 mg / kg, approximately 0.475 to approximately 1.700 mg / kg, approximately 0.475 to approximately 1.600 mg / kg, approximately 0.475 to approximately 1.500 mg / kg, approximately 0.475 to approximately 1.400 mg / kg, approximately 0.475 to approximately 1.400 mg / kg, approximately 0.475 to approximately 1,200 mg / kg, approximately 0.475 to approximately 1,100 mg / kg, approximately 0.475 to approximately 1,000 mg / kg, approximately 0.475 to approximately 0.900 mg / kg, approximately 0.475 to approximately 0.800 mg / kg, approximately 0.475 to approximately 0.700 mg / kg, approximately 0.475 to approximately 0.600 mg / kg, approximately 0.475 to approximately 0.500 mg / kg, approximately 0.875 to approximately 1.825 mg / kg, approximately 0.875 to approximately 1.800 mg / kg, Approximately 0.875 to approximately 1.700 mg / kg, approximately 0.875 to approximately 1.600 mg / kg, approximately 0.875 to approximately 1.500 mg / kg, approximately 0.875 to approximately 1.400 mg / kg, approximately 0.875 to approximately 1.400 mg / kg, approximately 0.875 to approximately 1.200 mg / kg, approximately 0.875 to approximately 1.100 mg / kg, approximately 0.875 to approximately 1.000 mg / kg, approximately 0.875 to approximately 0.900 mg / kg, approximately 0.900 to approximately 1.825 mg / kg, approximately 0.900 to 1.800 mg / kg, approximately 0.900 to 1.700 mg / kg, approximately 0.900 to 1.600 mg / kg, approximately 0.900 to 1.500 mg / kg, approximately 0.900 to 1.400 mg / kg, approximately 0.900 to 1.400 mg / kg, approximately 0.900 to 1.200 mg / kg, approximately 0.900 to 1.100 mg / kg, approximately 0.900 to 1.000 mg / kg, approximately 0.925 to 1.825 The drug is administered in doses of mg / kg, approximately 0.925 to approximately 1.800 mg / kg, approximately 0.925 to approximately 1.700 mg / kg, approximately 0.925 to approximately 1.600 mg / kg, approximately 0.925 to approximately 1.500 mg / kg, approximately 0.925 to approximately 1.400 mg / kg, approximately 0.925 to approximately 1.400 mg / kg, approximately 0.925 to approximately 1.200 mg / kg, approximately 0.925 to approximately 1.100 mg / kg, or approximately 0.925 to approximately 1.000 mg / kg. Intermediate values ​​and ranges of the aforementioned values ​​are also intended to be part of the present invention; for example, RNAi drugs can be administered to subjects in doses of approximately 0.015 mg / kg to approximately 0.45 mg / kg.

[0365] For example, RNAi drugs, for example, RNAi drugs in pharmaceutical compositions, are approximately 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg g, 0.325mg / kg, 0.35mg / kg, 0.375mg / kg, 0.4mg / kg, 0.425mg / kg, 0.45mg / kg, 0.475mg / kg, Approximately 0.5mg / kg, 0.525mg / kg, 0.55mg / kg, 0.575mg / kg, approximately 0.6mg / kg, 0.625mg / kg, 0.65mg / kg, 0.6 75mg / kg, about 0.7mg / kg, 0.725mg / kg, 0.75mg / kg, 0.775mg / kg, about 0.8mg / kg, 0.925mg / kg, 0.95m g / kg, 0.975mg / kg, approximately 1.0mg / kg, 1.025mg / kg, 1.05mg / kg, 1.075mg / kg, approximately 1.1mg / kg, 1.125mg / It can be administered in doses of kg, 1.15 mg / kg, 1.175 mg / kg, approximately 1.2 mg / kg, 1.225 mg / kg, 1.25 mg / kg, 1.275 mg / kg, approximately 1.3 mg / kg, 1.325 mg / kg, 1.35 mg / kg, 1.375 mg / kg, approximately 1.4 mg / kg, 1.425 mg / kg, 1.45 mg / kg, 1.475 mg / kg, approximately 1.5 mg / kg, 1.525 mg / kg, 1.55 mg / kg, 1.575 mg / kg, approximately 1.6 mg / kg, 1.625 mg / kg, 1.65 mg / kg, 1.675 mg / kg, approximately 1.7 mg / kg, 1.725 mg / kg, 1.75 mg / kg, 1.775 mg / kg, or approximately 1.8 mg / kg. It is intended that intermediate values ​​between the aforementioned values ​​are also part of the present invention.

[0366] Accordingly, in one embodiment, the present invention provides a method for preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression, for example, a bleeding disorder, for example, hemophilia, in a subject. The method comprises administering the iRNA agent of the present invention, for example, dsRNA (for example, a pharmaceutical composition comprising the dsRNA of the present invention) to a subject in a prophylactic effective dose, for example, a dose of about 0.200 mg / kg to about 1.825 mg / kg, thereby preventing at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0367] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression, for example, a bleeding disorder, for example, hemophilia, comprising administering to a subject, for example, a human, a therapeutically effective dose, for example, in the range of about 0.200 mg / kg to about 1.800 mg / kg, an iRNA agent targeting the Serpinc1 gene or a pharmaceutical composition comprising an iRNA agent targeting the Serpinc1 gene, to a subject, for example, a human, thereby providing a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0368] In another aspect, the present invention provides the use of the iRNA of the present invention, such as dsRNA, in a prophylactic effective dose, e.g., a dose ranging from about 0.200 mg / kg to about 1.825 mg / kg, to prevent at least one symptom in a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression.

[0369] In another aspect, the present invention provides the use of the iRNA agent of the present invention in a prophylactic effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, in the manufacture of an agent for preventing at least one symptom of a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression.

[0370] In another embodiment, the present invention provides the use of the iRNA agent of the present invention in therapeutically effective doses, e.g., doses ranging from about 0.200 mg / kg to about 1.825 mg / kg, for treating a subject, e.g., a subject that would benefit from reduced and / or inhibition of Serpinc1 expression.

[0371] In yet another aspect, the present invention relates to subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, such as subjects with bleeding disorders, such as hemophilia. The present invention provides for the use of a pharmaceutical composition containing an iRNA agent targeting the Serpinc1 gene, such as dsRNA, or an iRNA agent targeting the Serpinc1 gene in a therapeutically effective dose, such as from about 0.200 mg / kg to about 1.825 mg / kg, in the manufacture of a drug for treatment.

[0372] In some embodiments of the present invention, for example, if a double-stranded RNAi drug comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides different from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and not exceeding 3 nucleotides, and substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand to which it binds at the 3' end, then such a drug is administered in doses of about 0.200 to 1.825 mg / kg, for example, as doses of about 0.200 mg / kg to about 0.250 mg / kg; or as doses of about 0.425 mg / kg to about 0.475 mg / kg; or as doses of about 0.875 mg / kg to about 0.925 mg / kg; or as doses of about 1.775 mg / kg to about 1.825 mg / kg.

[0373] Accordingly, in one embodiment, the present invention provides a method for preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia, in a subject. The method involves administering a double-stranded ribonucleic acid (RNAi) agent (e.g., a pharmaceutical composition comprising an RNAi agent) to a subject in a prophylactic effective dose, e.g., from about 0.200 mg / kg to about 1.825 mg / kg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand bound at its 3' end, thereby preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression.

[0374] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression, for example, a bleeding disorder, for example, hemophilia, the method comprising administering to a subject, for example, a human, a pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent or an iRNA agent targeting the Serpinc1 gene, in a therapeutically effective dose, for example, from about 0.200 mg / kg to about 1.825 mg / kg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand bound at its 3' end, the double-stranded ribonucleic acid (RNAi) agent or an iRNA agent targeting the Serpinc1 gene, the method comprising administering to a subject, for example, a human, the method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0375] In another embodiment, the present invention relates to a double-stranded ribonucleic acid (RNAi) agent in a prophylactic effective dose, e.g., from about 0.200 mg / kg to about 1.825 mg / kg, for preventing at least one symptom of a subject suffering from a bleeding disorder, e.g., hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, and substantially all of the sense strand The use of double-stranded ribonucleic acid (RNAi) drugs is provided, where virtually all nucleotides in the nucleotide and antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end.

[0376] In a further embodiment, the present invention provides the use of a double-stranded ribonucleic acid (RNAi) agent in the manufacture of an agent for preventing at least one symptom of a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression, in a prophylactic effective dose, e.g., from about 0.200 mg / kg to about 1.825 mg / kg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end.

[0377] In another embodiment, the present invention provides the use of a double-stranded ribonucleic acid (RNAi) agent in a therapeutically effective dose, e.g., from about 0.200 mg / kg to about 1.825 mg / kg, for treating a subject, e.g., a subject that would benefit from reduced and / or inhibition of Serpinc1 expression, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end.

[0378] In yet another aspect, the present invention provides the use of a pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent, for example, a dsRNA or a double-stranded ribonucleic acid (RNAi) agent, in the manufacture of agents for treating subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, such as subjects with bleeding disorders, such as hemophilia. The agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end.

[0379] In some embodiments, the iRNA drug of the present invention is administered to subjects as a fixed dose. A “fixed dose” (e.g., a dose in mg units) means that a single dose of the iRNA drug is used for all subjects regardless of any specific subject-related factors such as body weight. In a particular embodiment, the fixed dose of the iRNA drug of the present invention is based on a given body weight or age.

[0380] In some embodiments, the RNAi drug is administered in doses ranging from approximately 25 mg to approximately 100 mg, for example, between approximately 25 mg and approximately 95 mg, between approximately 25 mg and approximately 90 mg, between approximately 25 mg and approximately 85 mg, between approximately 25 mg and approximately 80 mg, between approximately 25 mg and approximately 75 mg, between approximately 25 mg and approximately 70 mg, between approximately 25 mg and approximately 65 mg, between approximately 25 mg and approximately 60 mg, between approximately 25 mg and approximately 50 mg, between approximately 50 mg and approximately 100 mg, between approximately 50 mg and approximately 95 mg, between approximately 50 mg and approximately 90 mg, between approximately 50 mg and approximately 85 mg, between approximately 50 mg and approximately 80 mg, between approximately 30 mg and approximately 100 mg, or approximately 40 mg. It is administered as a fixed dose between approximately 90 mg, between approximately 40 mg and 80 mg, between approximately 60 mg and 100 mg, between approximately 60 mg and 90 mg, between approximately 25 mg and 55 mg, between approximately 25 mg and 65 mg, between approximately 30 mg and 95 mg, between approximately 30 mg and 85 mg, between approximately 30 mg and 75 mg, between approximately 30 mg and 65 mg, between approximately 30 mg and 55 mg, between approximately 40 mg and 95 mg, between approximately 40 mg and 85 mg, between approximately 40 mg and 75 mg, between approximately 40 mg and 65 mg, between approximately 40 mg and 55 mg, or between approximately 45 mg and 95 mg.

[0381] In some embodiments, the RNAi drug is administered as a fixed dose of approximately 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg.

[0382] Accordingly, in one embodiment, the present invention provides a method for preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression, e.g., bleeding disorders, e.g., hemophilia in a subject. The method comprises administering a fixed dose of the iRNA agent of the present invention, e.g., dsRNA (e.g., a pharmaceutical composition comprising the dsRNA of the present invention) to a subject, for a prophylactic effective dose, e.g., a fixed dose of about 25 mg to about 100 mg, thereby preventing at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the method comprises administering a fixed dose of the iRNA agent of the present invention, e.g., dsRNA (e.g., a pharmaceutical composition comprising the dsRNA of the present invention) to a subject, for a prophylactic effective dose, e.g., a fixed dose of about 50 mg, thereby preventing at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression. In another embodiment, the method comprises administering a prophylactic effective dose, for example, a fixed dose of about 80 mg of the iRNA agent of the present invention, for example, dsRNA (for example, a pharmaceutical composition comprising the dsRNA of the present invention), to a subject, thereby preventing at least one symptom of a disorder in which the subject would benefit from reduced Serpinc1 expression.

[0383] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression, for example, a bleeding disorder, for example, hemophilia, comprising administering to a subject, for example, a human, a fixed dose of a Serpinc1 gene-targeting iRNA agent or a pharmaceutical composition comprising a Serpinc1 gene-targeting iRNA agent, for example, about 25 mg to about 100 mg, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the method comprises administering to a subject a fixed dose of the iRNA agent of the present invention, for example, dsRNA (for example, a pharmaceutical composition comprising the dsRNA of the present invention), for example, about 50 mg, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression. In another embodiment, the method comprises administering to a subject a fixed dose of the iRNA agent of the present invention, for example, dsRNA (for example, a pharmaceutical composition comprising the dsRNA of the present invention), for example, about 80 mg, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0384] In another embodiment, the present invention provides the use of the iRNA of the present invention, e.g., dsRNA, in a fixed dose, e.g., about 25 mg to about 100 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibition of Serpinc1 expression, such as hemophilia. In one embodiment, the present invention provides the use of the iRNA of the present invention, e.g., dsRNA, in a fixed dose, e.g., about 50 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibition of Serpinc1 expression, such as hemophilia. In another embodiment, the present invention provides the use of the iRNA of the present invention, e.g., dsRNA, in a fixed dose, e.g., about 50 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibition of Serpinc1 expression, such as hemophilia. The present invention provides the use of iRNA, e.g., dsRNA, in a fixed dose, e.g., about 80 mg, for the prevention of at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited pinc1 expression.

[0385] In a further embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 25 mg to about 100 mg, in the manufacture of an agent for preventing at least one symptom in a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression. In one embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 50 mg, in the manufacture of an agent for preventing at least one symptom in a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression. In another embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 80 mg, in the manufacture of an agent for preventing at least one symptom in a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression.

[0386] In another embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 25 mg to about 100 mg, for treating a subject, for example, a subject that would benefit from reduced and / or inhibition of Serpinc1 expression. In one embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 50 mg, for treating a subject, for example, a subject that would benefit from reduced and / or inhibition of Serpinc1 expression. In another embodiment, the present invention provides the use of the iRNA agent of the present invention in a fixed dose, for example, about 80 mg, for treating a subject, for example, a subject that would benefit from reduced and / or inhibition of Serpinc1 expression.

[0387] In yet another embodiment, the present invention provides the use of a pharmaceutical composition comprising a Serpinc1 gene-targeting iRNA agent of the present invention, e.g., dsRNA, or a fixed dose of a Serpinc1 gene-targeting iRNA agent, such as a therapeutically effective dose, e.g., about 25 mg to about 100 mg, in the manufacture of a drug for treating a subject, e.g., a subject who would benefit from reduced and / or inhibition of Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia. In one embodiment, the present invention provides the use of a pharmaceutical composition comprising a Serpinc1 gene-targeting iRNA agent of the present invention, e.g., dsRNA, or a fixed dose of a Serpinc1 gene-targeting iRNA agent, such as a therapeutically effective dose, e.g., about 50 mg, in the manufacture of a drug for treating a subject, e.g., a subject who would benefit from reduced and / or inhibition of Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia. In another embodiment, the present invention provides the use of a pharmaceutical composition comprising a Serpinc1 gene-targeting iRNA agent of the present invention, such as dsRNA, or a fixed dose of a therapeutically effective dose, such as about 80 mg, of a Serpinc1 gene-targeting iRNA agent in the manufacture of a drug for treating subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, such as subjects with bleeding disorders, such as hemophilia.

[0388] In some embodiments of the present invention, for example, if a double-stranded RNAi drug comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides different not exceeding 3 nucleotides, and substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand to which it binds at the 3' end, then such a drug is available in fixed doses ranging from about 25 mg to about 100 mg, for example, as a fixed dose of about 25 mg; or as a fixed dose of about 50 mg; or as a fixed dose of about 80 mg. ; or administered as a fixed dose of approximately 100 mg. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0389] Accordingly, in one embodiment, the present invention provides a method for preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia, in a subject. The method involves administering a double-stranded ribonucleic acid (RNAi) agent (e.g., a pharmaceutical composition comprising an RNAi agent) to a subject in a fixed dose effective prophylactic dose, e.g., about 25 mg to about 100 mg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand bound at its 3' end, thereby preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0390] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia, the method comprising administering to a subject, e.g., a human, a pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent or an iRNA agent targeting the Serpinc1 gene, in a fixed dose therapeutically effective dose, e.g., about 25 mg to about 100 mg, comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand bound at its 3' end, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0391] In another embodiment, the present invention provides the use of a double-stranded ribonucleic acid (RNAi) agent in a fixed dose, e.g., about 25 mg to about 100 mg, for preventing at least one symptom of a subject suffering from a bleeding disorder, e.g., a disorder from which the subject would benefit from reduced and / or inhibition of Serpinc1 expression, such as hemophilia, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0392] In a further embodiment, the present invention relates to the manufacture of a double-stranded ribonucleic acid (RNAi) agent for the prevention of at least one symptom of a subject suffering from a bleeding disorder, such as hemophilia, which would benefit from reduced and / or inhibition of Serpinc1 expression, in a fixed dose prophylactic effective dose, e.g., about 25 mg to about 100 mg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, and substantially the sense strand This provides the use of a double-stranded ribonucleic acid (RNAi) drug in which all nucleotides and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0393] In another embodiment, the present invention provides the use of a double-stranded ribonucleic acid (RNAi) agent in a fixed dose therapeutically effective, e.g., about 25 mg to about 100 mg, for treating a subject, e.g., a subject that would benefit from reduced and / or inhibition of Serpinc1 expression, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0394] In yet another embodiment, the present invention provides the use of a pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent, for example, a dsRNA or a double-stranded ribonucleic acid (RNAi) agent, in the manufacture of agents for treating subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, such as subjects with bleeding disorders, such as hemophilia, etc., the subject being an iRNA agent of the present invention that targets the Serpinc1 gene, for example, a dsRNA or a double-stranded ribonucleic acid (RNAi) agent in a fixed dose, for example, about 25 mg to about 100 mg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region comprising the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides different not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0395] The methods and uses of the present invention are, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 The method includes administering the compositions described herein to reduce the expression of the target Serpinc1 gene for 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80 days. In one embodiment, the expression of the target Serpinc1 gene is reduced for a long duration, for example, at least about 7 days or more, for example, about 1 week, 2 weeks, 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, about a quarter, or longer.

[0396] A decrease in gene expression can be assessed by any method known in the art. For example, a decrease in Serpinc1 expression can be determined by methods common to those skilled in the art, such as determining the mRNA expression level of Serpinc1 using Northern blotting, qRT-PCR, determining the protein level of Serpinc1 using methods common to those skilled in the art, such as Western blotting, immunological methods, and / or by confirming the biological activity of Serpinc1 that affects one or more molecules (or blood coagulation itself in an in vivo environment) related to the cellular blood coagulation mechanism. In one embodiment, to assess Serpinc1 expression, for example, ROTEM® Thromboelast of whole blood is used. Mechanism analysis is used to determine thrombin formation time, clot formation time, and / or coagulation time.

[0397] Administration of dsRNA by the method and use of the present invention may result in a reduction of the severity, signs, symptoms, and / or markers of such disease or disorder in patients with Serpinc1-related disease. In this context, “reduction” means a statistically significant decrease of such a level. The reduction may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0398] The effectiveness of treatment or prevention of a disease can be assessed, for example, by measuring the levels of disease progression, disease remission, symptom severity, bleeding frequency, pain relief, quality of life, the dosage of medication required to sustain the effect of the treatment, disease markers, or any other measurable parameters appropriate to a given treated disease or disease targeted for prevention. Monitoring the effectiveness of treatment or prevention by measuring any one of such parameters, or any combination of parameters, is well within the capabilities of a person skilled in the art. For example, the effectiveness of treatment for bleeding disorders can be assessed, for example, by regularly monitoring thrombin:antithrombin levels. Comparison of later measurements with initial measurements provides a physician with an indication of whether the treatment is effective. Monitoring the effectiveness of treatment or prevention by measuring any one of such parameters, or any combination of parameters, is well within the capabilities of a person skilled in the art. In relation to the administration of Serpinc1-targeting iRNA or its pharmaceutical composition, "effective against" bleeding disorders means that administration in a clinically appropriate manner results in beneficial effects, such as improvement of symptoms, recovery, alleviation of disease, extension of life, improvement of quality of life, or other effects that are generally recognized as positive by physicians familiar with the treatment and associated causes of bleeding disorders, in at least a statistically significant proportion of patients.

[0399] The effectiveness of a treatment or preventive measure is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening of symptoms or the onset of symptoms that would otherwise be expected. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or more, in the measurable parameters of the disease indicates an effective treatment. The effectiveness of a given iRNA drug or a formulation of that drug can also be determined using experimental animal models for a given disease known in the art. When using experimental animal models, the effectiveness of a treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

[0400] Alternatively, effectiveness can be assessed by a reduction in disease severity as determined by a person skilled in the art based on a clinically recognized disease severity assessment scale. For example, any positive change resulting from a reduction in disease severity as determined using an appropriate scale represents appropriate treatment with the iRNA or iRNA preparation described herein.

[0401] iRNA (or a pharmaceutical composition containing iRNA) can be administered to the subject approximately once a week, approximately twice a month, approximately once every six weeks, approximately once every two months, or once a quarter.

[0402] Double-stranded iRNA drugs can be administered to subjects in one or more doses. For example, double-stranded iRNA drugs can be administered to subjects in a monthly dose of approximately 0.200 mg / kg to approximately 1.825 mg / kg. Alternatively, double-stranded iRNA drugs can be administered to subjects in a fixed dose of approximately 25 mg to approximately 100 mg.

[0403] In one embodiment, the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. The double-stranded RNAi drug is administered to the subject as a once-monthly dose of approximately 0.200 mg / kg to approximately 0.250 mg / kg, for example, approximately 0.225 mg / kg.

[0404] In another embodiment, the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. The double-stranded RNAi drug is administered to the subject as a once-monthly dose of approximately 0.425 mg / kg to approximately 0.475 mg / kg, for example, approximately 0.450 mg / kg.

[0405] In yet another embodiment, the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end, and the double-stranded RNAi drug is administered to the subject as a once-monthly dose of about 0.875 mg / kg to about 0.925 mg / kg, for example, about 0.900 mg / kg.

[0406] In one embodiment, the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. The double-stranded RNAi drug is administered to the subject in a once-monthly dose of approximately 1.775 mg / kg to approximately 1.825 mg / kg, for example, approximately 1.800 mg / kg.

[0407] In one embodiment, the double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. The double-stranded RNAi drug is administered to a subject as a fixed dose of about 25 to about 100 mg, for example, about 25 mg, 50 mg, 80 mg, or 100 mg. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0408] Administration can be repeated on a regular basis, for example, once a month for one, two, three, four months, or longer periods. After the initial treatment regimen, it is also possible to administer treatment on a fewer frequency basis, for example, once a month for three months. Subsequently, the treatment can be repeated quarterly for a period of one year or longer.

[0409] Therefore, in some embodiments, the RNAi drug is administered in a dosing regimen that includes a "loading phase" of short-interval dosing, followed by a "maintenance phase" in which the RNAi drug is administered at longer intervals.

[0410] Loading and / or maintenance schedules may be repeated in one or more iterations, depending on the circumstances. The number of iterations may also depend on the achievement of the desired effect, e.g., suppression of the Serpinc1 gene, and / or the achievement of therapeutic or prophylactic effects, e.g., increased blood coagulation, reduced clot formation time, and / or reduced clotting time.

[0411] iRNA administration raises Serpinc1 levels in the patient's cells, tissues, blood, urine, or other compartments to at least approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, and 49%. It can be reduced by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0412] iRNA can be administered by intravenous infusion over a period of time, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or approximately 25 minutes.

[0413] Before administering the full dose of iRNA, a smaller dose, such as a 5% infusion, may be given to the patient, and adverse effects such as allergic reactions may be monitored. In another example, the patient may be monitored for undesirable immunostimulatory effects, such as increased cytokine levels (e.g., TNF-alpha or INF-alpha).

[0414] Due to their inhibitory effect on Serpinc1 expression, the compositions according to the present invention or pharmaceutical compositions prepared therefrom can improve the quality of life.

[0415] The iRNA of the present invention can be administered in a “naked” form or as “free iRNA.” Naked iRNA is administered in the absence of the pharmaceutical composition. It may also be naked iRNA in a suitable buffer. The buffer may contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS). The pH and molar osmotic concentration of the buffer containing the iRNA can be adjusted to suit administration to the subject.

[0416] Alternatively, the iRNA of the present invention can be administered as a pharmaceutical composition such as a dsRNA liposome formulation.

[0417] Subjects who would benefit from reduced and / or inhibited Serpinc1 gene expression are those with bleeding disorders as described herein, e.g., hereditary bleeding disorders or acquired bleeding disorders. In one embodiment, subjects with hereditary bleeding disorders include hemophilia, e.g., hemophilia. The subject has disease A, B, or C. In one embodiment, a subject with a hereditary bleeding disorder, e.g., hemophilia, is an inhibitor subject (a subject that has become resistant to replacement clotting factors). In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C. Treatment of a subject who would benefit from reduced and / or inhibition of Serpinc1 gene expression includes therapeutic treatment (e.g., on-demand, e.g., the subject is bleeding (spontaneous bleeding or bleeding as a result of trauma) and not clotting) and prophylactic treatment (e.g., the subject is not bleeding and / or undergoes surgery).

[0418] The present invention further provides methods and uses for the use of iRNA or a pharmaceutical composition thereof to treat subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, for example, subjects having bleeding disorders, in combination with other pharmaceuticals and / or other therapeutic methods, for example, known pharmaceuticals and / or known therapeutic methods, such as those currently used to treat these disorders.

[0419] For example, in certain embodiments, iRNAs targeting Serpinc1 are administered in combination with agents useful in treating bleeding disorders, for example, as described elsewhere in this specification. For example, suitable additional therapeutic agents and treatments for patients who may benefit from reduced Serpinc1 expression, such as those with bleeding disorders, include fresh frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrate; virus-inactivated vWF-containing FVIII concentrate; desensitization therapy, sometimes including high doses of FVIII or FIX with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasma exchange therapy with or without antifibrinolytic therapy in combination with immunosuppression and FVIII or FIX infusion; immune tolerance induction (ITI) with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and / or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytic agents such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilia agents; corticosteroids; immunosuppressants; and estrogens.

[0420] iRNA and additional therapeutic agents and / or treatments may be administered simultaneously and / or in the same combination, for example, parenterally, or the additional therapeutic agents may be administered as part of separate compositions, at separate times, and / or by other methods known in the art or described herein.

[0421] In one embodiment, the present invention provides compound AD-57213 (sense chain: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and antisense in a once-monthly dose of approximately 0.200 mg / kg to approximately 1.800 mg / kg, for example, approximately 0.200 mg / kg to approximately 0.250 mg / kg; approximately 0.425 mg / kg to approximately 0.475 mg / kg; approximately 0.875 mg / kg to approximately 0.925 mg / kg; or approximately 1.775 mg / kg to approximately 1.825 mg / kg) The chain: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage) is provided as a method for treating subjects with bleeding disorders, such as hemophilia, by subcutaneous administration.

[0422] In another embodiment, the present invention provides compound AD-57213 (sense chain: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfu) in fixed doses ranging from about 25 mg to about 100 mg, for example, about 25 mg, about 50 mg, about 80 mg, or about 100 mg. The present invention provides a method for treating subjects with bleeding disorders, such as hemophilia, by subcutaneous administration of CfaAf-3' (SEQ ID NO: 13) and antisense chain: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate linkage). In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0423] III. iRNA for use in the method of the present invention This specification describes methods for using improved double-stranded RNAi agents that inhibit the expression of the Serpinc1 gene in cells, such as those in mammals, such as those in humans with Serpinc1-related disorders, such as hemorrhagic disorders, such as hemophilia.

[0424] Accordingly, the present invention provides a double-stranded RNAi agent having chemical modifications that can inhibit the expression of a target gene (i.e., the Serpinc1 gene) in vivo. In certain embodiments of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention that is "substantially all nucleotides modified" is not completely modified but is mostly modified and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0425] RNAi drugs include a sense strand and an antisense strand. Each strand of an RNAi drug may be in the range of 12 to 30 nucleotides in length. For example, each strand may be between 14 to 30 nucleotides, 17 to 30 nucleotides, 19 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or between 21 and 23 nucleotides.

[0426] The sense and antisense strands typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "RNAi drug." The double-stranded region of the RNAi drug can be 12–30 nucleotide pairs long. For example, the double-stranded region may be between 14–30 nucleotide pairs, 17–30 nucleotide pairs, 27–30 nucleotide pairs, 17–23 nucleotide pairs, 17–21 nucleotide pairs, 17–19 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 19–21 nucleotide pairs, 21–25 nucleotide pairs, or between 21–23 nucleotide pairs. In another example, the double-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths.

[0427] In one embodiment, the RNAi drug may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhangs may result from one strand being longer than the other, or from two strands of the same length being misaligned. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be a different sequence. The first and second strands may also contain additional bases, or other non-basic linkers, for example, forming a hairpin. It is joined by -.

[0428] In one embodiment, the nucleotides in the overhang region of the RNAi drug may be modified or unmodified nucleotides, each independently and not limited to, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof, including 2'-saccharide modifications. For example, TT may be an overhang sequence at the end of either strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be a different sequence.

[0429] The 5'- or 3'-overhangs on the sense strand, antisense strand, or both strands of an RNAi drug are phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between them, and the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located on the antisense strand. In one embodiment, this 3'-overhang is located on the sense strand.

[0430] RNAi drugs may contain only a single overhang that can enhance the buffering activity of RNAi without affecting its overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand, or conversely, at the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Typically, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While we do not wish to be constrained by theory, asymmetric blunt ends at the 5' end and 3' end overhangs of the antisense strand are advantageous for introducing guide strands into RISC processes.

[0431] Any nucleic acids addressed in the present invention are synthesized and / or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linking) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse linking, etc.); base modifications, such as substitution with stable bases, unstable bases, or bases that base-pair with a wide range of partners, base removal (debastic nucleotide), or conjugate bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or skeletal modifications, including modifications or substitutions of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain a modified skeleton or do not contain natural internucleoside linkages. RNAs with modified skeletons include those that do not have a phosphorus atom in their skeleton. For the purposes of this specification and as is sometimes mentioned in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton are also considered to be oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside skeleton.

[0432] Examples of modified RNA backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and 3'-alkylene phosphonates, as well as other alkyl phosphonates, chiral phosphonates, phosphinates, 3'-aminophosphoamides, and aminoalkyl Phosphoramides containing thiophosphoramides, thionophosphoramides, thionoalkylphosphonates, thionoalkylphosphotryesters, and boranophosphates having normal 3'-5' linkages, their 2'-5' linked analogues, and those with reverse polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0433] Representative U.S. patents teaching the preparation of the phosphorus-containing linkage described above include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717. ;No. 5,321,131;No. 5,399,676;No. 5,405,939;No. 5,453,496;No. 5,455,233;No. 5,466,677;No. 5,476,9 No. 25; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,58 No. 7,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6 ,239,265;No.6,277,603;No.6,326,199;No.6,346,614;No.6,444,423;No.6,531,590;No.6,534,639 Examples include Patent No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of these are incorporated herein by reference.

[0434] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include skeletons with morpholino linkages (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamic acid skeletons; methyleneimino and methylenehydrazino skeletons; sulfonic acid and sulfonamide skeletons; amide skeletons; and other skeletons having mixed N, O, S, and CH2 components.

[0435] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; and 5 Nos. 489,677; Nos. 5,541,307; Nos. 5,561,225; Nos. 5,596,086; Nos. 5,602,240; Nos. 5,608,046; Nos. 5,610,289; Nos. 5,618,704; Nos. 5,623,070; Nos. 5,663,312; Nos. 5,633,360; Nos. 5,677,437; and Nos. 5,677,439, the entire contents of each of these are incorporated herein by reference.

[0436] In other embodiments, suitable RNA mimetic bodies are considered for use in iRNA, where both sugar and nucleoside linkages, i.e., the nucleotide unit backbone, are replaced with novel groups. The base unit is maintained for hybridization with suitable nucleic acid target compounds. Such have been shown to have excellent hybridization properties. RNA mimetic compounds, a type of oligomeric compound, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleic acid bases are retained and directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262, the entire contents of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, pp. 1497-1500.

[0437] Some embodiments of the present invention include RNA having a phosphorothioate skeleton and oligonucleosides having a heteroatom skeleton, in particular the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2--[known as methylene(methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2--[wherein the natural phosphodiester skeleton is represented as --O--P--O--CH2--], as well as the amide skeleton of the above-mentioned U.S. Patent No. 5,602,240. In some embodiments, the RNA discussed herein has the morpholino skeleton structure of the above-mentioned U.S. Patent No. 5,034,506.

[0438] Modified RNA may also contain one or more substituted sugar moieties. iRNAs discussed herein, such as dsRNAs, may contain at the 2' position one of the following:OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10They can be alkenyls and alkynyls. Exemplarily preferred modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n The formula contains CH3)2, where n and m range from 1 to about 10. In other embodiments, the dsDNA is below the 2' position:C1~C 10 The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups that enhance the pharmacokinetic properties of iRNA, or groups that improve the pharmacodynamic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the following examples herein.

[0439] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications occur at other positions in the iRNA's RNA, particularly at the 3' position of the sugar in the 3'-terminal nucleotide or 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. iRNA may also have sugar mimetic molecules such as a cyclobutyl moiety instead of pentofuranosyl sugar. Representative U.S. patents teaching the preparation of modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and 5,567. Examples include Nos. 811; Nos. 5,576,427; Nos. 5,591,722; Nos. 5,597,909; Nos. 5,610,300; Nos. 5,627,053; Nos. 5,639,873; Nos. 5,646,265; Nos. 5,658,873; Nos. 5,670,633; and Nos. 5,700,920, some of which are owned by the same person as the present application. The entire contents of each of the above are incorporated herein by reference.

[0440] iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil ( This includes pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as other synthetic and native nucleic acid bases such as 3-deazaguanine and 3-deazaadenine.Further examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; Englisch et al., Angewandte Chemie, International Edition, 1991, pp. 30, 613; and Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and it is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0441] Representative U.S. patents teaching the preparation of the above-mentioned modified nucleic acid bases and other modified nucleic acid bases include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; and 5,367. ,066;No.5,432,272;No.5,457,187;No.5,459,255;No.5,484,908;No.5,502,177;No.5,525,711;No.5,552, No. 540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,8 This includes Nos. 86; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of these are incorporated herein by reference.

[0442] The RNA of iRNA is also modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge that connects two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention include the RNA of iRNA and are also modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in the 3'-end structural conformation. It has been shown that adding locked nucleic acids to siRNA increases the stability of siRNA in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): pp. 439-447; Mook, O. et al., (2007) Mol Canc Ther 6(3): pp. 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0443] Examples of bicyclic nucleosides for use in polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides containing a 4'-2' bridge. Examples of such 4'-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogues; U.S. Examples include: 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, for example, U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, pp. 118-134); and 4'-CH2-C(=CH2)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the above are incorporated herein by reference.

[0444] Further representative U.S. patents and U.S. patent application publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; and 7,034,133. Examples include Patent Nos. 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; and U.S. Patent Application Publications 2008 / 0039618 and 2009 / 0012281, the entire contents of which are incorporated herein by reference.

[0445] For example, any of the aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see International Publication No. 99 / 14226).

[0446] The RNA of the iRNA can also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety with a 4'-CH(CH3)-0-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S conformation referred to herein as “S-cEt”.

[0447] The iRNA of the present invention may also include one or more “conformational restriction nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs fix the ribose ring to a stable conformation and increase hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal position for stability and affinity, and reduces distortion of the ribose ring.

[0448] Representative publications teaching some of the above-mentioned preparations of the CRN include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication International Publication No. 2013 / 036868, the entire contents of which are incorporated herein by reference.

[0449] One or more nucleotides of the iRNA of the present invention may also include a hydroxymethyl-substituted nucleotide. A “hydroxymethyl-substituted nucleotide” is an acyclic 2'-3'-seco-nucleotide also known as an “unlocked nucleic acid” (“UNA”) modification.

[0450] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0451] Potentially stable modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3''-phosphate, and reverse base dT (idT). Disclosure of these modifications can be found in PCT publication number International Publication No. 2011 / 005861.

[0452] A. Modified iRNA containing the motif of the present invention In particular aspects of the present invention, the double-stranded RNAi agents of the present invention are incorporated herein by reference, for example, in their entirety, as described herein by reference in the United States Patent Application Filed November 18, 2011. This includes agents having chemical modifications disclosed in Provisional Patent Application No. 61 / 561,710, or PCT / U.S. Patent Application Publication No. 2012 / 065691, filed on November 16, 2012.

[0453] As described herein, in U.S. Provisional Patent Application No. 61 / 561,710, and PCT / U.S. Patent Application Publication No. 2012 / 065691, better results are obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and / or antisense strands of the RNAi drug, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi drug are otherwise fully modified. The introduction of these motifs disrupts the modification pattern of the sense and / or antisense strands, if present. The RNAi drug is optionally conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi drug exhibits superior gene silencing activity.

[0454] More specifically, it was surprisingly discovered that the gene silencing activity of the RNAi drug was significantly enhanced when the sense and antisense strands of the double-stranded RNAi drug were modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi drug.

[0455] In one embodiment, the RNAi drug is a 19-nucleotide blunt-ended double-stranded drug. It is a bluntmer, and the sense strand has three consecutive nuclei from the 5' end at positions 7, 8, and 9. The rheotide contains at least one motif of three 2'-F modifications. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end to positions 11, 12, and 13.

[0456] In another embodiment, the RNAi drug is a 20-nucleotide blunt-ended double strand, where the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides from the 5' end at positions 8, 9, and 10. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0457] In yet another embodiment, the RNAi drug is a 21-nucleotide blunt-ended double strand, where the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides from the 5' end at positions 9, 10, and 11. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0458] In one embodiment, the RNAi drug comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi drug being blunt and the other end containing two nucleotide overhangs. Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand. When the two nucleotide overhangs are at the 3' end of the antisense strand, there are two phosphorothioate nucleotide linkages between the three terminal nucleotides, with two of the three nucleotides being overhang nucleotides and the third nucleotide being a paired nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi drug further has two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In this embodiment, all nucleotides in the sense and antisense strands of the RNAi drug, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in alternating motifs. Optionally, the RNAi drug further comprises a ligand (preferably GalNAc3).

[0459] In one embodiment, the RNAi drug comprises a sense strand and an antisense strand, and the RNAi drug comprises a first strand having a nucleotide length of at least 25 and 29 or less, and a second strand having a nucleotide length of 30 or less, and containing at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides long, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand so that when the RNAi drug is introduced into mammalian cells, it reduces the expression of the target gene, and dicer cleavage of the RNAi drug preferentially yields siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi drug further comprises a ligand.

[0460] In one embodiment, the sense strand of the RNAi drug contains at least one motif of three identical modifications in three consecutive nucleotides, one of which is located at a cleavage site on the sense strand.

[0461] In one embodiment, the antisense strand of the RNAi drug may also contain at least one motif of three identical modifications in three consecutive nucleotides, one of which is located at or near the cleavage site of the antisense strand.

[0462] In RNAi drugs with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0463] The sense strand of an RNAi drug may contain at least one motif of three identical modifications in three consecutive nucleotides at the cleavage site of the strand; the antisense strand may have at least one motif of three identical modifications in three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may be aligned such that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide duplication, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0464] In one embodiment, the sense strand of an RNAi drug may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motifs may be wing modifications. This is possible. The term "wing modification" as used herein refers to a wing modification at or near the break site of the same chain. A winged modification refers to a motif located on a different part of the chain, separated from the chief. Winged modifications are either adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are directly adjacent to each other, their chemical structures are different; if they are separated by one or more nucleotides, their chemical structures may be the same or different. More than one winged modification can exist. For example, if two winged modifications exist, each winged modification may be located at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0465] Similar to the sense strand, the antisense strand of an RNAi drug may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those present in the sense strand.

[0466] In one embodiment, the wing modification in the sense or antisense strand of the RNAi drug typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0467] In another embodiment, the wing modification in the sense or antisense strand of the RNAi drug typically does not contain the first one or two paired nucleotides within the 3' end, 5' end, or double-stranded regions at both ends of the strand.

[0468] If the sense strand and antisense strand of an RNAi drug each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0469] If the sense strand and antisense strand of an RNAi drug each contain at least two wing modifications, the sense strand and antisense strand are aligned such that: two modifications from one strand each are located at one end of the double-stranded region and have one, two, or three nucleotide duplicates; two modifications from one strand each are located at the other end of the double-stranded region and have one, two, or three nucleotide duplicates; and two modifications from one strand are located on each side of the read motif and have one, two, or three nucleotide duplicates in the double-stranded region.

[0470] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi drug, including nucleotides that are part of a motif, are modified. Each nucleotide is modified with the same or different modifications, which may include one or more changes to one or more unbound phosphate oxygens and / or bound phosphate oxygens, or both; changes to components of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety by a "dephosphorylating" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0471] Because nucleic acids are polymers of subunits, many modifications, such as modifications to bases, or phosphate moieties, or unbound oxygen atoms in phosphate moieties, are located at repeating positions within the nucleic acid. In some cases, modifications are present at all desired positions in the nucleic acid, but often this is not the case. For example, modifications may be present only at the 3' or 5' terminal position, or only in the terminal region, e.g., at the terminal nucleotides, or at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications may be present only in the double-stranded region of RNA, or in RNA It may be present only in the single-stranded region. For example, phosphorothioate modifications at the unbound O position may be present only on one or both ends, in the terminal region, e.g., on the terminal nucleotide or only on the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in both the double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends are phosphorylated.

[0472] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang are modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modifications instead of ribosaccharides in nucleic acid bases, and modifications of phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0473] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0474] At least two distinct modifications are typically present on the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0475] In one embodiment, Na and / or N b This includes alternating pattern modifications. As used herein, the term “alternating motif” means a motif having one or more modifications, where each modification is present in alternating nucleotides on a single chain. Alternating nucleotides may refer to one modification every other nucleotide, one modification every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motifs may be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0476] The types of modifications contained in alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0477] In one embodiment, the RNAi agent of the present invention includes an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift may be such that the modifying groups of the nucleotides in the sense strand correspond to differently modified groups of the nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motif in the sense strand may start from "ABABAB" from 5' to 3' of the strand. In the antisense chain, the alternating motif begins with "BABABA" from 5' to 3' of the chain within the double-stranded region. Alternatively, the alternating motif in the sense chain may begin with "AABBAABB" from 5' to 3' of the chain, and the alternating motif in the antisense chain may begin with "BBAABBAA" from 5' to 3' of the chain within the double-stranded region, thereby resulting in a complete or partial shift of the modification pattern between the sense and antisense chains.

[0478] In one embodiment, the RNAi drug comprises a pattern of alternating 2'-O-methyl and 2'-F modifications in the sense strand, with a shift in the pattern of alternating 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., a 2'-O-methyl modified nucleotide in the sense strand base-pairs with a 2'-F modified nucleotide in the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0479] Introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand disrupts the initial modification pattern present in the sense strand and / or antisense strand. This disruption of the modification pattern in the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand unexpectedly enhances gene silencing activity against the target gene.

[0480] In one embodiment, when three identical modification motifs on three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, a portion of the sequence containing the motif is "···N a YYYN b..." where "Y" represents the modification of three identical modification motifs in three consecutive nucleotides, and "N a " and "N b " represents a modification of a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and N a and N b These can be the same or different modifications. Or, N a and / or N b This may or may not exist if a wing modifier is present.

[0481] RNAi agents may further contain at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present on any nucleotide in the sense strand, the antisense strand, or both strands at any position in the chain. For example, the internucleotide linkage modification may be present on all nucleotides in the sense strand and / or antisense strand; each internucleotide linkage modification may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of internucleotide linkage modifications in the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications in the antisense strand.

[0482] In one embodiment, the RNAi includes a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. The internucleotide linkage modification is also formed to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked by a phosphorothioate or methylphosphonate internucleotide linkage. Furthermore, there may be additional phosphorothioate or methylphosphonate nucleotide linkages that connect the overhang nucleotide to the adjacent paired nucleotide. For example, there may be at least two phosphorothioate nucleotide linkages between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0483] In one embodiment, two nucleotide overhangs are located at the 3' end of the antisense strand, with two phosphorothioate nucleotide linkages between the three terminal nucleotides, two of which are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the RNAi drug may further have two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0484] In one embodiment, the RNAi agent includes mismatches with the target, mismatches within the double helix, or combinations thereof. A “mismatch” may be a non-canonical base pairing or a nucleotide pairing other than a canonical one. Mismatches may occur in overhang regions or double helix regions. Base pairs are evaluated based on their tendency to promote dissociation or melting (e.g., with respect to the free energy of binding or dissociation of a particular pair, the simplest method being to examine each individual pair, although similar or equivalent analyses are also used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere in this specification), are preferred over canonical (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over canonical pairings. A "universal base" is a base that exhibits the ability to substitute any of the four normal bases (G, C, A, and U) without significantly destabilizing neighboring base-pair interactions or destroying the expected functional biochemical availability of the modified oligonucleotide. Non-limiting examples of universal bases include 2'-deoxyinosine (hypoxanthine deoxynucleotide) or its derivatives, nitroazole analogs, and hydrophobic aromatic non-hydrogen-bonded bases.

[0485] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the groups A:U, G:U, and I:C, and a mismatch pair to facilitate the dissociation of the antisense strand at the 5' end of the double helix, e.g., a non-canonical or non-canonical pair or a pair containing a universal base.

[0486] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0487] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In yet another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of two dT nucleotides at the 3' ends of the deoxythymine nucleotides, e.g., the sense strand and / or antisense strand.

[0488] In one embodiment, the sense strand sequence is given by formula (I): 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) (In the formula: i and j are independently either 0 or 1; p and q are independently between 0 and 6; each N a However, each sequence independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two differently modified nucleotides; each N b However, each n independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; p and n q However, they independently represent overhang nucleotides; Here, Nb and Y do not have the same modification; (XXX, YYY, and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides.) This is represented by [formula]. Preferably, all YYY nucleotides are 2'-F modified nucleotides.

[0489] In one embodiment, N a and / or N b This includes alternating pattern modifications.

[0490] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, if the RNAi drug has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be located at or near the sense strand cleavage site, counting from the first nucleotide from the 5' end; or, optionally, from the first paired nucleotide in the double-stranded region from the 5' end (e.g., it may be located at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0491] In one embodiment, i is 1 and J is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain is given by the following equation: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (1b); 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q It is represented by 3' (Id).

[0492] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0493] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0494] When the sense strand is represented by formula (Id), each N b independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0495] Each of X, Y, and Z may be the same as or different from each other.

[0496] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5’ n p -N a -YYY-N a -n q 3’ (Ia) represented by

[0497] When the sense strand is represented by formula (Ia), each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0498] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (II) (In the formula: k and l are either 0 or 1 independently; p' and q' are independently between 0 and 6; each N a ' represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, with each sequence containing at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and n q ' represents an overhanging nucleotide independently; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications in three consecutive nucleotides. It is represented by [this].

[0499] In one embodiment, N a 'and / or N b ' includes alternating pattern modifications.

[0500] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if an RNAi drug has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is located at positions 11, 12, 13.

[0501] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0502] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0503] Therefore, the antisense chain is given by the following formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb); 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc); or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId) It is represented by [this].

[0504] If the antisense chain is represented by equation (IIb), then N b' contains oligonucleotides with modified values ​​of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Represents the nucleotide sequence. Each N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently.

[0505] If the antisense chain is expressed as equation (IIc), then N b ' represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently.

[0506] If the antisense chain is expressed as equation (IId), then each N b ' represents an oligonucleotide sequence containing independently modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0507] In another embodiment, k is 0, l is 0, and the antisense chain is given by: 5' n p’ -Na ’ -Y'Y'Y'-Na ’ -nq ’ 3' (Ia) It is represented by [this].

[0508] If the antisense chain is represented by equation (IIa), then each N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently.

[0509] Each of X', Y', and Z' can be the same as or different from one another.

[0510] Each nucleotide in the sense and antisense strands can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0511] In one embodiment, the sense strand of the RNAi drug may contain YYY motifs located at positions 9, 10, and 11 of the strand, starting from the first nucleotide from the 5' end, or optionally from the 5' end, starting from the first paired nucleotide in the double-stranded region; where Y represents a 2'-F modification. The sense strand may further contain XXX or ZZZ motifs as wing modifications at the opposite end of the double-stranded region; where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0512] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, starting counting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0513] A sense chain represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense chain represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0514] Therefore, the RNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double helix is ​​given by formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q ' 5 ' (III) (In the formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, where each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, Each n may or may not exist. p ',n p , n q ', and n q However, they independently represent overhang nucleotides; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' are represented by (each independently representing a single motif of three identical modifications on three consecutive nucleotides).

[0515] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0516] An example combination of sense and antisense strands that form an RNAi double helix is ​​given by the following formula: 5' n p -N a -YYY-N a -n q 3' 3' n p '-N a '-Y'Y'Y'-N a 'n q ' 5 ' (IIIa) 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' 3' n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q ' 5 ' (IIIb) 5' n p -N a -XXX-N b -YYY-N a -n q 3' 3' n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q ' 5 ' (IIIc) 5' n p -N a -XXX-N b -YYY-Nb -ZZZ-N a -n q 3' 3' n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q ' 5 ' (IIId) 5' -N a -YYY-N a - 3' 3' n p '-N a '-Y'Y'Y'-N a ' 5 ' (IIIe) Includes.

[0517] If an RNAi drug is represented by formula (IIIa), then each N a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0518] If an RNAi drug is represented by formula (IIIb), then each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0519] When an RNAi drug is represented by formula (IIIc), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0520] When an RNAi drug is represented by formula (IIId), each N b , Nb ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently. a , N a ', N b , and N b Each of these contains an independent alternating pattern of modification.

[0521] When an RNAi drug is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently. a , N a ', N b , and N b Each of these contains an independent alternating pattern of modification.

[0522] When an RNAi drug is represented by formula (IIIe), each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides independently, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides.

[0523] In equations (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), each of X, Y, and Z may be the same as or different from one another.

[0524] If an RNAi drug is represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), then at least one Y nucleotide may base-pair with one of the Y' nucleotides; or at least two Y nucleotides may base-pair with the corresponding Y' nucleotides; or all three Y nucleotides may base-pair with the corresponding Y' nucleotides.

[0525] If an RNAi drug is represented by formula (IIIb) or (IIId), then at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form a base pair with the corresponding Z' nucleotide; or all three Z nucleotides may form a base pair with the corresponding Z' nucleotide.

[0526] If an RNAi drug is represented by formula (IIIc) or (IIId), then at least one of the X nucleotides may base-pair with one of the X' nucleotides; or at least two of the X nucleotides may base-pair with the corresponding X' nucleotides; or all three of the X nucleotides may base-pair with the corresponding X' nucleotides.

[0527] In one embodiment, the modification on the Y nucleotide differs from the modification on the Y' nucleotide, Z Modifications on a nucleotide are different from modifications on a Z' nucleotide, and / or modifications on an X nucleotide are different from modifications on an X' nucleotide.

[0528] In one embodiment, if the RNAi drug is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi drug is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p' is linked to an adjacent nucleotide via phosphorothioate linkage. In yet another embodiment, if the RNAi drug is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. In another embodiment, if the RNAi drug is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker.

[0529] In one embodiment, if the RNAi drug is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker.

[0530] In one embodiment, the RNAi drug is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), which are linked by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further contains ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

[0531] In one embodiment, the RNAi drug is a multimer containing three, four, five, six or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), where the double helixes are linked by linkers. The linkers may be cleavable or incleavable. The multimer may further contain ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

[0532] In one embodiment, two RNAi drugs represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) are ligated together at one or both of their 5' and 3' ends and optionally conjugated to a ligand. Each drug may target the same gene or two different genes; or each drug may target the same gene at two different target sites.

[0533] Various publications describe the multimeric RNAi drugs used in the method of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Publication No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520, the entire contents of each of these publications are incorporated herein by reference.

[0534] RNAi drugs containing conjugations of one or more carbohydrate moieties can optimize one or more properties of the RNAi drug. Often, the carbohydrate moiety is conjugated to a modified subunit of the RNAi drug. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA drug is replaced by a non-carbohydrate (preferably cyclic) carrier to which another moiety, such as a carbohydrate ligand, is conjugated. Ribonucleotide subunits in which the ribose sugar of the subunit is thus substituted are referred to herein as ribose-substituted modified subunits (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier may be a monocyclic system, or may contain two or more rings, such as a fused ring. The cyclic carrier may be a fully saturated ring system, or may contain one or more double bonds.

[0535] The ligand is conjugated with the polynucleotide via a carrier. The carrier comprises (i) at least one “skeletal attachment point,” preferably two “skeletal attachment points,” and (ii) at least one “tethering attachment point.” As used herein, “skeletal attachment point” refers to a bond suitable for incorporating the carrier into the ribonucleic acid skeleton, which may contain a functional group, e.g., a hydroxyl group, or generally a skeleton, e.g., a phosphate or modified phosphate, e.g., sulfur. In some embodiments, a “tethering attachment point” (TAP) refers to a ring atom of the cyclic carrier, e.g., a carbon atom or heteroatom (different from the atom providing the skeletal attachment points), that connects a selected portion. This portion may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the selected portion is connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often contains a functional group, e.g., an amino group, or generally provides a bond suitable for incorporating another chemical component, e.g., a ligand, into the ring or for tethering.

[0536] RNAi drugs may be conjugated with ligands via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is pyrrolidinyl, pyrazolinil, pyrazolidinyl, imidazolinil, imidazolidinyl, piperidinil, piperazinyl, [1,3]dioxolane, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinyl, tetrazolinil The acyclic group is selected from trahydrofuryl and decalin; preferably, the acyclic group is selected from the selinol skeleton or the diethanolamine skeleton.

[0537] In certain embodiments, the RNAi agent for use in the method of the present invention is AD-57213 (sense strand: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and antisense strand: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate linkage).

[0538] These drugs may further contain ligands.

[0539] Ligand The double-stranded RNA (dsRNA) agent of the present invention is optionally conjugated with one or more ligands. The ligand is conjugated to the sense strand, antisense strand, or both strands at its 3' end, 5' end, or both ends. For example, the ligand is conjugated to the sense strand. In a preferred embodiment, the ligand is conjugated to the 3' end of the sense strand.

[0540] In one embodiment, the ligand is a carbohydrate conjugate such as a monosaccharide. In one embodiment, the ligand is N-acetylgalactosamine (GalNAc), GalNAc, or a GalNAc derivative. In certain embodiments of the present invention, GalNAc or a GalNAc derivative binds to the iRNA drug of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative binds to the iRNA drug of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA drug of the present invention via a trivalent linker.

[0541] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is

[0542] [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0543] In one embodiment, GalNAc or a GalNAc derivative is GalNAc3: [ka] That is the case.

[0544] In some embodiments, a ligand, such as a GalNAc ligand, binds to the 3' end of the RNAi drug. In one embodiment, the RNAi drug is shown in the following diagram. [ka] (In the formula, X is either O or S) As shown, it is conjugated with a ligand, such as a GalNAc ligand. In one embodiment, X is O.

[0545] Various substances can bind to the RNAi drug of the present invention. Preferred components are ligands that bind directly or indirectly, preferably by covalent bond, via an intervening tether.

[0546] In a preferred embodiment, the ligand alters the distribution, targeting, or lifespan of the molecule into which it is incorporated. In a preferred embodiment, the ligand provides, for example, enhanced affinity to selected targets, such as molecules, cells or cell types, compartments, receptors, such as compartments of cells or organs, tissues, organs, or regions of the body, compared to species without such ligand. A ligand that provides enhanced affinity to selected targets is also called a targeted ligand.

[0547] Some ligands may possess endosomal lysis properties. Endosomal lysis ligands promote the lysis of endosomes and / or the transport of the composition of the present invention, or its components, from endosomes to the cytoplasm of cells. Endosomal lysis ligands may be polyanionic peptides or peptide mimetic compounds exhibiting pH-dependent membrane activity and membrane fusion properties. In one embodiment, the endosomal lysis ligand is presumed to adopt its active conformation at the pH of endosomes. The "active" conformation is the conformation in which the endosomal lysis ligand promotes the lysis of endosomes and / or the transport of the composition of the present invention, or its components, from endosomes to the cytoplasm of cells. Exemplary endosomal lysis ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptide (Vogel et al., J.Am.Chem.Soc., 1996, 118:1581-1586), and their derivatives (Turk Examples include et al., Biochem. Biophys. Acta, 2002, 1559: pp. 56-68. In one embodiment, the endosomal lysate may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomal lysate may be linear or branched.

[0548] Ligands can enhance transport, hybridization, and specificity properties, and may also improve the nuclease resistance of polymer molecules containing the resulting natural or modified oligoribonucleotides, or any combination of monomers and / or natural or modified ribonucleotides described herein.

[0549] Ligands can generally include, for example, therapeutic modifiers to enhance uptake; for example, diagnostic compounds or reporter groups to monitor distribution; crosslinking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetic compounds.

[0550] Ligands may include proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or natural substances such as lipids. Ligands may also be recombinant or synthetic molecules such as synthetic polymers, e.g., synthetic polyamino acids, or oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0551] Ligands may also include target groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, and antibodies that bind to specific cell types, such as kidney cells. Target groups may include thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, RGD peptides, RGD peptide mimes, or aptamers.

[0552] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, or chelating agents (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O Examples include 3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ tetraaza macrocyclic complex), dinitrophenyl, HRP, or AP.

[0553] The ligand is a protein, such as a glycoprotein or a peptide, such as a corrigan. The ligand may be a molecule that has specific affinity for antibodies, such as antibodies that bind to a particular cell type, such as cancer cells, endothelial cells, or osteocytes. The ligand may also include hormones and hormone receptors. The ligand may also include lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, or non-peptide species such as aptamers. The ligand may be, for example, a lipopolysaccharide, a p38MAP kinase activator, or an NF-κB activator.

[0554] A ligand can be a substance, such as a drug, that can enhance the uptake of an iRNA drug into a cell, for example, by disrupting the cytoskeleton, such as by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0555] Ligands can increase the uptake of oligonucleotides into cells, for example, by activating inflammatory responses. Exemplary ligands that may have such effects include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon.

[0556] In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA.

[0557] Lipid-based ligands can be used to modulate, for example, control the binding of conjugates to target tissues. For instance, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to target the kidney and therefore less likely to be removed from the body. A lipid or lipid-based ligand that does not bind as strongly to HSA can be used to ensure that the conjugate targets the kidney.

[0558] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate is dispersed preferably in non-renal tissue. However, this affinity is preferably not strong enough so that the HSA-ligand bond is not reversed.

[0559] In another preferred embodiment, the lipid-based ligand either weakly or not binds to HSA at all, so the conjugate is preferably dispersed in the kidney. Other parts that target kidney cells can also be used instead of, or in addition to, the lipid-based ligand.

[0560] In another embodiment, the ligand is a portion taken up by target cells, such as proliferating cells, e.g., a vitamin. These are particularly useful for treating disorders characterized by undesirable cell proliferation, such as malignant or non-malignant cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include: Examples include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. Also included are HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL).

[0561] In another embodiment, the ligand is a cell permeabilizer, preferably a helix-type cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or Antennapedia. If the agent is a peptide, it can be modified, including the use of peptidyl mimes, inversion isomers, non-peptide or pseudopeptide linkages, and D-amino acids. Preferably, this helix-type agent is an alpha-helix-type agent having a lipophilic phase and an oleophobic phase.

[0562] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The peptide moiety or peptide mimetic moiety may have an amino acid length of about 5 to 50, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (for example, composed mainly of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a restricting peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogues containing a hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) may also be the targeting moiety. The peptide portion may be a “delivery” peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to function as delivery peptides. Peptides or peptide mimetic molecules, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries, are encoded by random sequences of DNA (Lam et al., Nature, 354: pp. 82-84, 1991). Preferably, peptides or peptide mimetic molecules linked to iRNA drugs via incorporated monomer units are cell-targeting peptides such as arginine-glycine-aspartate (RGD)-peptides or RGD mimetic molecules. The peptide portion may range in length from about 5 amino acids to about 40 amino acids. The peptide portion may undergo structural modifications, such as those to enhance stability or direct conformational properties.Any of the following structural modifications can ...

Claims

1. A method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression, comprising administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to the subject, ranging from about 25 mg to about 100 mg. The double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, The method thereby prevents at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression.

2. A method for treating a subject with a disorder that would benefit from reduced Serpinc1 expression, comprising administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to the subject, ranging from about 25 mg to about 100 mg. The double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, The method for treating a subject having a disorder that would thereby benefit from reduced Serpinc1 expression.

3. The method according to claim 1 or 2, wherein a fixed dose of a double-stranded RNAi agent is administered to the subject once a month, once every six weeks, once every two months, or four times a year.

4. The method according to claim 1 or 2, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 25 mg.

5. The method according to claim 1 or 2, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 50 mg.

6. The method according to claim 1 or 2, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 80 mg.

7. The method according to claim 1 or 2, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 100 mg.

8. The method according to any one of claims 4 to 7, wherein the double-stranded RNAi drug is administered subcutaneously to the subject.

9. The method according to any one of claims 1 to 8, wherein administration of a double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%.

10. The method according to any one of claims 1 to 9, wherein administration of a double-stranded RNAi agent to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a non-impaired subject that would benefit from reduced Serpinc1 expression.

11. The method according to any one of claims 1 to 10, wherein administration of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administration of factor VIII to the subject.

12. The method according to any one of claims 1 to 11, wherein administration of a double-stranded RNAi agent to a subject is sufficient to achieve a peak thrombin formation level of more than approximately 40% in the subject.

13. The method according to any one of claims 1 to 12, wherein administration of a double-stranded RNAi agent to a subject has a disorder that would benefit from reduced Serpinc1 expression, and the administration of the double-stranded RNAi agent reduces the subject's on-demand annual bleeding rate (ABR) by approximately 80 to approximately 95 percent compared to the median ABR of a subject that has not received the double-stranded RNAi agent.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. The method according to any one of claims 1 to 14, wherein the disorder is a hemorrhagic disorder.

16. The method according to claim 15, wherein the bleeding disorder is an acquired bleeding disorder or a hereditary bleeding disorder.

17. The method according to claim 15, wherein the bleeding disorder is hemophilia.

18. The method according to claim 17, wherein the hemophilia is hemophilia A, hemophilia B, or hemophilia C; the hemophilia is hemophilia A and the subject is an inhibitor subject; the hemophilia is hemophilia B and the subject is an inhibitor subject; or the hemophilia is hemophilia C and the subject is an inhibitor subject.

19. The method according to any one of claims 1 to 18, wherein the double-stranded RNAi drug is administered subcutaneously to the subject.

20. The method according to any one of claims 1 to 19, wherein all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

21. The method according to any one of claims 1 to 20, wherein the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases including nucleotides.

22. The method according to any one of claims 1 to 21, wherein the complementary region is at least 17 nucleotides long.

23. The method according to any one of claims 1 to 22, wherein the complementary region has a nucleotide length between 19 and 21.

24. The method according to claim 23, wherein the complementary region is 19 nucleotides long.

25. The method according to any one of claims 1 to 24, wherein each chain does not exceed 30 nucleotides in length.

26. At least one strand contains a 3' overhang of at least one nucleotide, claim The method described in any one of items 1 to 25.

27. The method according to any one of claims 1 to 26, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

28. The method according to any one of claims 1 to 27, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

29. The ligand is 【Chemistry 1】 The method according to any one of claims 1 to 28.

30. Double-stranded RNAi drugs conjugate with ligands as shown in the schematic diagram below. 【Chemistry 2】 The method according to claim 29, wherein X is O or S.

31. The method according to claim 30, wherein X is O.

32. The method according to any one of claims 1 to 31, wherein the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

33. The double-stranded RNAi drug consists of a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisequence strand containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15). The method according to any one of claims 1 to 32, comprising a lance chain.

34. The sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), The method according to claim 33, wherein A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

35. The sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage. The sense chain is conjugated with a ligand as shown in the schematic diagram below, 【Transformation 3】 The method according to any one of claims 1 to 34, wherein X is O or S.

36. The method according to any one of claims 1 to 35, wherein the double-stranded RNAi drug is administered to a subject as a pharmaceutical composition.

37. The method according to claim 36, wherein a double-stranded RNAi drug in a non-buffered solution is administered.

38. The method according to claim 37, wherein the non-buffer is saline solution or water.

39. The method according to claim 36, wherein the double-stranded RNAi drug is administered to the subject together with a buffer.

40. The method according to claim 39, wherein the buffer comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

41. The method according to claim 40, wherein the buffer solution is phosphate-buffered saline (PBS).

42. A kit for carrying out the method described in any one of claims 1 to 41, a) Double-stranded RNAi drugs, b) Instructions for use, c) Depending on the circumstances, means for administering the double-stranded RNAi drug to the target The kit includes the above.

43. A method for preventing at least one symptom of a disorder in a subject who would benefit from reduced Serpinc1 expression, comprising administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent in a dose of approximately 40 mg to approximately 90 mg to the subject, The double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, The method thereby prevents at least one symptom of a subject having a disorder that would benefit from reduced Serpinc1 expression.

44. A method for treating a subject with a disorder that would benefit from reduced Serpinc1 expression, comprising administering a fixed dose of a double-stranded ribonucleic acid (RNAi) agent to the subject at a dose of approximately 40 mg to approximately 90 mg, The double-stranded RNAi drug comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) and at least 15 consecutive nucleotides not exceeding 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand conjugates with a ligand bound at its 3' end, The method for treating a subject having a disorder that would thereby benefit from reduced Serpinc1 expression.

45. The method according to claim 43 or 44, wherein a fixed dose of a double-stranded RNAi agent is administered to the subject once a month, once every six weeks, once every two months, or four times a year.

46. The method according to claim 43 or 44, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 25 mg.

47. The method according to claim 43 or 44, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 50 mg.

48. The method according to claim 43 or 44, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 80 mg.

49. The method according to claim 43 or 44, wherein the double-stranded RNAi drug is administered to the subject as a fixed dose of approximately 100 mg.

50. The method according to any one of claims 46 to 49, wherein the double-stranded RNAi drug is administered subcutaneously to the subject.

51. The method according to any one of claims 43 to 50, wherein administration of a double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by approximately 70% to approximately 95%.

52. The method according to any one of claims 43 to 51, wherein administration of a double-stranded RNAi agent to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in a non-impaired subject that would benefit from reduced Serpinc1 expression.

53. The method according to any one of claims 43 to 52, wherein administration of a double-stranded RNAi agent to a subject is sufficient to bring the peak thrombin formation level in the subject to approximately the same level as that achieved by administration of factor VIII to the subject.

54. The method according to any one of claims 43 to 53, wherein administration of a double-stranded RNAi agent to a subject is sufficient to achieve a peak thrombin formation level of more than approximately 40% in the subject.

55. The method according to any one of claims 43 to 54, wherein administration of a double-stranded RNAi agent to a subject has a disorder that would benefit from reduced Serpinc1 expression, and the administration of the double-stranded RNAi agent reduces the subject's on-demand annual bleeding rate (ABR) by about 80 to about 95 percent compared to the median ABR of a subject that has not received the double-stranded RNAi agent.

56. The method according to any one of claims 43 to 55, wherein the subject is a human.

57. The method according to any one of claims 43 to 56, wherein the disorder is a hemorrhagic disorder.

58. The method according to claim 57, wherein the bleeding disorder is an acquired bleeding disorder or a hereditary bleeding disorder.

59. The method according to claim 57, wherein the bleeding disorder is hemophilia.

60. The method according to claim 59, wherein the hemophilia is hemophilia A, hemophilia B, or hemophilia C; the hemophilia is hemophilia A and the subject is an inhibitor subject; the hemophilia is hemophilia B and the subject is an inhibitor subject; or the hemophilia is hemophilia C and the subject is an inhibitor subject.

61. The method according to any one of claims 43 to 60, wherein the double-stranded RNAi drug is administered subcutaneously to the subject.

62. The method according to any one of claims 43 to 61, wherein all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

63. The method according to any one of claims 43 to 62, wherein the modified nucleotide is independently selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases including nucleotides.

64. The method according to any one of claims 43 to 63, wherein the complementary region is at least 17 nucleotides long.

65. The method according to any one of claims 43 to 64, wherein the complementary region has a nucleotide length between 19 and 21.

66. The method according to claim 65, wherein the complementary region is 19 nucleotides long.

67. The method according to any one of claims 43 to 66, wherein each chain is not more than 30 nucleotides long.

68. The method according to any one of claims 43 to 67, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

69. The method according to any one of claims 43 to 68, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

70. The method according to any one of claims 43 to 69, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

71. The ligand is 【Chemistry 4】 The method according to any one of claims 43 to 70.

72. Double-stranded RNAi drugs conjugate with ligands as shown in the schematic diagram below. 【Transformation 5】 The method according to claim 71, wherein X is O or S.

73. The method according to claim 72, wherein X is O.

74. The method according to any one of claims 43 to 31, wherein the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

75. The method according to any one of claims 43 to 74, wherein the double-stranded RNAi drug comprises a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand containing the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

76. The sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), The method according to claim 75, wherein A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

77. The sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage. The sense chain is conjugated with a ligand as shown in the schematic diagram below, 【Transformation 6】 The method according to any one of claims 43 to 76, wherein X is O or S.

78. The method according to any one of claims 43 to 77, wherein the double-stranded RNAi drug is administered to a subject as a pharmaceutical composition.

79. The method according to claim 78, wherein a double-stranded RNAi drug in a non-buffered solution is administered.

80. The method according to claim 79, wherein the non-buffer is saline solution or water.

81. The method according to claim 78, wherein the double-stranded RNAi drug is administered to the subject together with a buffer.

82. The method according to claim 81, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

83. The method according to claim 82, wherein the buffer solution is phosphate-buffered saline (PBS).

84. A kit for carrying out the method described in any one of claims 43 to 83, a) Double-stranded RNAi drugs, b) Instructions for use, c) Depending on the circumstances, means for administering the double-stranded RNAi drug to the target The kit includes the above.

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